Centrifugally separating samples in a container having a seal and containing a plunger for opening the seal

ABSTRACT

A separation container for extracting a portion of a sample for use or testing and method for preparing samples for downstream use or testing are provided. The separation container may include a body defining an internal chamber. The body may define an opening, and the body may be configured to receive the sample within the internal chamber. The separation container may further include a seal disposed across the opening, such that the seal may be configured to seal the opening of the body, and a plunger movably disposed at least partially inside the internal chamber. The plunger may be configured to be actuated to open the seal and express the portion of the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/537,731 entitled “Microbial Isolation Tube” and filed Jul. 27, 2017,and this application claims the benefit of U.S. Provisional ApplicationNo. 62/643,918 entitled “Microbial Isolation Tube” and filed Mar. 16,2018. Each of the foregoing applications is hereby incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to a separation container and associatedsystems and methods for separating a sample via centrifugation andextracting a portion of the sample for use or testing.

BACKGROUND OF THE INVENTION

Sample preparation devices may typically separate a microorganism from asurrounding sample material (e.g., a blood sample) by centrifugation.Traditionally these systems require separate lysing, washing, decanting,and spinning steps, often requiring repeated washing, decanting, andspinning of the sample until the final, concentrated microorganism isobtained. Most, if not all, of these steps required separate userhandling and different containers and equipment to perform.

Moreover, due to the wide variety of possible microorganisms that may betested in the same separation container, the final properties of theconcentrated microorganism (e.g., the density, viscosity, mass, etc.)may be difficult to predict. Many existing devices and methods requiredelicate handling and a precise application of force to transfer themicroorganism into other testing apparatus after centrifugation. Theseprocesses are heavily dependent on user training and experience toobtain accurate, precise results. In addition, when handling dangerousmicroorganisms from sample material, it is often preferred to minimizehuman interaction with the sample as much as possible.

The inventors have identified a number of additional deficiencies andproblems associated with conventional microbial separation products andother associated systems and methods. Through applied effort, ingenuity,and innovation, many of these identified problems have been solved bydeveloping solutions that are included in embodiments of the presentinvention, many examples of which are described in detail herein.

BRIEF SUMMARY

Embodiments of the present invention herein include separationcontainers, centrifugation assemblies, and associated methods andsystems for separating a sample via centrifugation and extracting aportion of the sample for use or testing. In some embodiments, aseparation container may be provided for separating a sample viacentrifugation and extracting a portion of the sample for use ortesting. The separation container may include a body defining aninternal chamber. The body may define an opening at a first end, and thebody may be configured to receive the sample within the internalchamber. The separation container may further include a seal disposedacross the opening, such that the seal may be configured to seal theopening of the body. The separation container may also include a plungermovably disposed at least partially inside the internal chamber. Theplunger may be configured to be actuated to open the seal and extractthe portion of the sample.

The body may define an axis extending from the first end to a secondend. A longitudinal member of the plunger may be disposed on the axis.In some embodiments, the internal chamber may define a diameter radialto the axis, and the diameter may narrow from a collection diameter to apellet diameter in a direction extending axially from the second end tothe first end. At least a portion of the plunger may be configured tosealingly engage the body at a portion of the body corresponding to thepellet diameter. The at least the portion of the plunger may define aplunger diameter radial to a length of the longitudinal member, and theplunger diameter may be greater than the pellet diameter, and may definean interference fit between the plunger and the pellet region. In someembodiments, the plunger diameter may less than the collection diameter.The at least the portion of the plunger may include a sealing ribdisposed circumferentially about the longitudinal member of the plunger,and the sealing rib may be configured to engage the body at the portionof the body corresponding to the pellet diameter.

In some embodiments, the plunger may be configured to allow the portionof the sample to pass by the plunger from a second end towards the firstend during centrifugation, and the plunger may be configured to preventa remaining part of the sample from traveling to the first end duringactuation of the plunger, such that during actuation, the plunger maydivide the internal chamber into two sub-chambers.

In some embodiments, the plunger may be buoyant in water or a densitycushion material. In some further embodiments, the plunger may define aspecific density of 0.95 or less relative to the water or the densitycushion material. The plunger may further define a specific density of0.9 or less relative to the water or the density cushion material. Insome embodiments, the plunger may be buoyant in a mixture of the waterand the density cushion material.

In some embodiments, the plunger may include one or more sealing ribsnear a distal end that engage a narrow, pellet region of the separationcontainer to seal the pellet region and apply a pressure to themicroorganism sample. A blade or other pointed region of the plunger maythen pierce the seal of the separation container from within thepressurized pellet region to express the microorganism from theseparation container under pressure. The sealing rib(s) may preventcontamination of the sample by sealing the remaining fluid above theplunger from the microorganism sample, while also ensuring that thepellet is completely expressed from the pellet region.

In some embodiments, the plunger may define a point at a first distalend of a longitudinal member of the plunger, and the point may beconfigured to pierce the seal at the first opening to allow fluidcommunication between the internal chamber and an area outside the bodyvia the opening.

The separation container may further include a flexible sealing memberdisposed at a second end of the body, and a second distal end of theplunger may be configured to extend at least partially into the flexiblesealing member, such that compression of the flexible sealing member mayactuate the plunger. The separation container may further include a capsecured to the body at the second end, and a portion of the flexiblesealing member may be configured to be disposed between the cap and thebody, and the cap may define an opening through which a second portionof the flexible sealing member and the second distal end of the plungermay be configured to extend. In some embodiments, the flexible sealingmember may include a bellows gasket defining an open end configured toreceive a portion of the plunger therein. The bellows gasket may furtherdefine a closed end configured to seal the internal chamber of the body.

In some embodiments, the separation container may include a samplecollecting vessel configured to removably engage the body. The samplecollecting vessel may be configured to surround the opening, such thatthe sample collecting vessel may be configured to collect the portion ofthe sample passing through the seal. The sample collecting vessel maycontain fluids to facilitate the resuspension, testing, and/or growth ofcells recovered from the sample portion.

The separation container may also include a rheological control memberdisposed in the internal chamber of the body. The rheological controlmember may define a barrier configured to reduce mixing of the sampleand a density cushion. In some embodiments, the barrier of therheological control member may define an annular structure disposedabout the plunger. In some embodiments, the rheological control membermay be buoyant in water and in a density cushion material.

In some embodiments, the seal may include a membrane, and in someembodiments, the membrane may include a foil sheet.

In another embodiment, a centrifugation assembly may be provided. Thecentrifugation assembly may include a separation container forseparating a sample via centrifugation and extracting a portion of thesample for use or testing. In such embodiments, the separation containermay include body defining an internal chamber. The body may define anopening at a first end, and the body may be configured to receive thesample within the internal chamber for centrifugation. The separationcontainer of the centrifugation assembly may further include a sealdisposed across the opening, such that the seal may be configured toseal the opening of the body. The separation container may also includea plunger movably disposed at least partially inside the internalchamber, and the plunger may be configured to be actuated to open theseal and extract the portion of the sample. The centrifuge assembly mayalso include a centrifuge cup configured to receive the separationcontainer. The centrifuge cup may include a side wall configured to abutthe body, and a bottom wall configured to abut the first end of thebody. The bottom wall may be configured to support the seal of theseparation container during centrifugation.

In yet another embodiment, a method for preparing viable and/ornon-viable portions of a sample for testing may be provided. The methodmay include disposing the sample portion into a separation container.The separation container may include a body defining an internalchamber, and the body may define an opening at a first end. Theseparation container may further include a seal disposed across theopening, such that the seal may be configured to seal the opening of thebody. The separation container may also include a plunger movablydisposed at least partially inside the internal chamber, and the plungermay be configured to be actuated to open the seal. The separationcontainer may further include a density cushion disposed in the internalchamber of the body. The method may include centrifuging the separationcontainer to create a pellet from a portion of the sample within theinternal chamber, and expressing the pellet from the opening in the bodyby depressing the plunger.

In some embodiments of the method, centrifuging the separation containerto create the pellet may include allowing the portion of the sample topass the plunger and collect at the first end of the body. Expressingthe pellet may include depressing the plunger into sealing engagementwith a portion of the body to create pressure between the plunger andthe seal, and expelling the pellet from the opening under the pressureby opening the seal.

In some embodiments, the pellet may include viable portions of thesample suitable for a culture step.

In some embodiments, the pellet may include viable portions of thesample suitable for antibiotic susceptibility testing (AST) andphenotypic identification methods.

In some embodiments, the pellet may include portions of the samplesuitable for identification by mass spectrometry (e.g. MALDI-TOF).

In some embodiments, the pellet may include portions of the samplesuitable for other applications such as nucleic acid amplificationtechniques, spectroscopy techniques (e.g., Raman, FTIR), immunoassaytechniques, probe-based assays, agglutination tests etc.).

In some embodiments, a rheological control member may be used that mayseal between the plunger and the wall of the body to prevent mixing ofthe density cushion and the sample. In such embodiments, the rheologicalcontrol member may be released by the wall when the wall expandsoutwardly during centrifugation.

In another embodiment, a separation container may be provided forseparating a sample via centrifugation and extracting a portion of thesample for use or testing. The separation container may include a bodydefining an internal chamber. The body may include a wall at leastpartially bounding the internal chamber. The body may include an openingat a first end, and the body may define an axis extending from the firstend to a second end. In some embodiments, an internal chamber defines adiameter radial to the axis, and the wall may be at least partiallyflexible such that the diameter of the internal chamber is a firstdiameter in a static state and the diameter of the internal chamber mayexpand to a second diameter during centrifugation. The body may beconfigured to receive the sample within the internal chamber. Theseparation container may further include a seal disposed across theopening, such that the seal may be configured to seal the opening of thebody, and a plunger movably disposed at least partially inside theinternal chamber. A longitudinal member of the plunger may be disposedon the axis of the body. The plunger may be configured to be actuated toopen the seal and extract a portion of the sample. The separationcontainer may further include a rheological control member disposed inthe internal chamber. The rheological control member may define a borethrough which the longitudinal member of the plunger is disposed, suchthat the rheological control member may be disposed between thelongitudinal member and the wall. In some embodiments, the rheologicalcontrol member may define an outermost diameter radial to the axis ofthe body. The outermost diameter of the rheological control member maybe greater than the first diameter, and the second diameter may begreater than the outermost diameter of the rheological control member.

In some embodiments, the body may include a collection region definingthe diameter. The body may include a widened region defining a greaterdiameter than the diameter of the collection region, and the greaterdiameter of the widened region may be greater than the outermostdiameter of the rheological control member.

The wall may include an annular shoulder at which the diameter of theinternal chamber changes. The first diameter may be defined on a narrowside of the annular shoulder in a static state, and the annular shouldermay be configured to engage the rheological control member.

In some embodiments, the rheological control member may include a secondannular shoulder comprising a wide side defining the outermost diameterand a narrow side.

The separation container may include a gasket disposed circumferentiallyabout the longitudinal member, and the gasket may be configured to sealan opening between the bore of the rheological control member and theplunger.

In another embodiment, a separation container and end cap assembly maybe provided for separating a sample via centrifugation and extracting aportion of the sample for use or testing. The assembly may include abody, a seal, an end cap, and a plunger. The body may define an internalchamber, and the body may define an opening at a first end. The body maybe configured to receive the sample within the internal chamber. Theseal may be disposed across the opening, such that the seal may beconfigured to seal the opening of the body. The end cap may be at thefirst end, and the end cap may be attachable to the body. In someembodiments, the seal may be disposed between the end cap and the body.The plunger may be movably disposed at least partially inside theinternal chamber, and the plunger may be configured to be actuated toopen the seal and extract a portion of the sample.

In another embodiment, a separation container may be provided forseparating a sample via centrifugation and extracting a portion of thesample for use or testing. The separation container may include a body,a seal, a plunger, and a flexible sealing member. The body may includean internal chamber, and the body may define a first opening at a firstend and a second opening at a second end. The body may be configured toreceive the sample within the internal chamber. In some embodiments, theseal may be disposed across the opening, such that the seal may beconfigured to seal the opening of the body. The plunger may be disposedat least partially inside the internal chamber, and the plunger may beconfigured to be actuated to open the seal and extract a portion of thesample. The flexible sealing member may at least partially cover thesecond opening. At least a portion of the plunger may be configured toextend at least partially into the flexible sealing member, such thatcompression of the flexible sealing member may actuate the plunger.

In some embodiments, the flexible sealing member may define a wallconfigured to at least partially surround the portion of the plunger.The wall may define an inwardly concave shape, such that the wall may beconfigured to flex outwardly from the plunger when the plunger isactuated. The flexible sealing member may include a firstcircumferential wall segment connected to a top of the flexible sealingmember, a second circumferential wall segment connected to the firstcircumferential wall segment, and a third circumferential wall segmentconnected to the second circumferential wall segment. The secondcircumferential wall segment may be concentric about a longitudinal axisof the plunger. The first circumferential wall segment and the secondcircumferential wall segment may each be angled at least partiallyinwardly towards the plunger from their respective connections to thesecond circumferential wall segment.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Having thus described the disclosure in general terms, reference willnow be made to the accompanying drawings, which are not drawn to scale,and wherein:

FIG. 1 shows an exploded view of a separation container according tosome embodiments discussed herein;

FIG. 2 shows a side elevation view of the separation container of FIG.1;

FIG. 3 shows a cross-sectional view of FIG. 2 taken along Section A-A;

FIG. 4 shows a top plan view of the separation container of FIG. 1;

FIG. 5 shows another side elevation view of the separation container ofFIG. 1 showing the plunger in a raised position and the seal intact;

FIG. 6 shows a cross-sectional view of the separation container of FIG.5;

FIG. 7 shows a detail view of the pellet region and first distal end ofthe plunger of Detail B in FIG. 5;

FIG. 8 shows a side elevation view of the separation container of FIG. 1showing the plunger in a depressed position and the seal being opened;

FIG. 9 shows a cross-sectional view of the separation container of FIG.8;

FIG. 10 shows a top plan view of the separation container of FIG. 8;

FIG. 11 shows another side elevation view of the separation container ofFIG. 8;

FIG. 12 shows a cross-sectional view of FIG. 11 taken along Section B-B;

FIG. 13 shows a detail view of the pellet region and first distal end ofthe plunger of Detail A in FIG. 12;

FIG. 14 shows an exploded view of a separation container according towith some embodiments discussed herein;

FIG. 15 shows a side elevation view of the separation container of FIG.14 showing the plunger in a raised position and the seal intact;

FIG. 16 shows a cross-sectional view of the separation container of FIG.15 taken along Section A-A;

FIG. 17 shows a top plan view of the separation container of FIG. 14;

FIG. 18 shows another side elevation view of the separation container ofFIG. 14 showing the plunger in a raised position;

FIG. 19 shows a cross-sectional view of the separation container of FIG.18 taken along Section B-B;

FIG. 20 shows another cross-sectional view of the separation containerof FIG. 18 taken along Section B-B;

FIG. 21 shows a detail view of the rheological control member of DetailA in FIG. 20;

FIG. 22 shows a detail view of the pellet region and first distal end ofthe plunger of Detail B in FIG. 19;

FIG. 23-26 show various views of the barrier of the rheological controlmember of the separation container of FIG. 14;

FIG. 27-31 show various views of the adaptor of the separation containerof FIG. 14;

FIG. 32 shows an exploded view of a separation container according tosome embodiments discussed herein;

FIG. 33 shows a side elevation view of the separation container of FIG.32 showing the plunger in a raised position and the seal intact;

FIG. 34 shows a cross-sectional view of the separation container of FIG.33 taken along Section A-A;

FIG. 35 shows a top plan view of the separation container of FIG. 32;

FIG. 36 shows another side elevation view of the separation container ofFIG. 32 showing the plunger in a raised position;

FIG. 37 shows a cross-sectional view of the separation container of FIG.36 taken along Section B-B;

FIG. 38 shows a detail view of the pellet region and first distal end ofthe plunger of Detail B in FIG. 37;

FIG. 39 shows a side elevation view of a body and cap of the separationcontainer of FIG. 32;

FIG. 40 shows a cross-sectional view of the body, cap, and rheologicalcontrol member of FIG. 39 taken along Section A-A;

FIG. 41 shows a top plan view of the body, cap, and rheological controlmember of FIG. 40;

FIG. 42 shows a side elevation view of a body and cap of the separationcontainer of FIG. 32;

FIG. 43 shows a cross-sectional view of the body, cap, and rheologicalcontrol member of FIG. 42 taken along Section B-B;

FIG. 44 shows a detail view of the first end of the body of Detail A inFIG. 43;

FIG. 45 shows a cross-sectional view of the rheological control memberof FIG. 32;

FIG. 46 shows a side elevation view of the rheological control member ofFIG. 32;

FIG. 47 shows another cross-sectional view of the rheological controlmember of FIG. 32;

FIG. 48 shows an exploded view of the rheological member of FIG. 32;

FIG. 49 shows a perspective, cross-sectional view of the separationcontainer of FIG. 32;

FIG. 50 shows an exploded view of a separation container according tosome embodiments discussed herein;

FIG. 51 shows a side elevation view of the separation container of FIG.50 showing the plunger in a raised position and the seal intact;

FIG. 52 shows a cross-sectional view of the separation container of FIG.51 taken along Section A-A;

FIG. 53 shows a top plan view of the separation container of FIG. 50;

FIG. 54 shows another side elevation view of the separation container ofFIG. 50 showing the plunger in a raised position;

FIG. 55 shows a cross-sectional view of the separation container of FIG.54 taken along Section B-B;

FIG. 56 shows a detail view of the pellet region and first distal end ofthe plunger of Detail B in FIG. 55;

FIG. 57-67 show various views of a plunger 110 according to someembodiments discussed herein, including a perspective view (FIG. 57);side views (FIGS. 58, 64); detail views (FIGS. 59-60, 63, 66-67); a topview (FIG. 61); a bottom view (FIG. 62); and a downward cross-sectionalview (FIG. 65);

FIG. 68 shows an exploded view of a separation container according tosome embodiments discussed herein;

FIG. 69 shows a side elevation view of the separation container of FIG.68 showing the plunger in a raised position and the seal intact;

FIG. 70 shows a cross-sectional view of the separation container of FIG.69 taken along Section A-A;

FIG. 71 shows a top plan view of the separation container of FIG. 68;

FIG. 72 shows a detail view of the pellet region and first distal end ofthe plunger of Detail A of FIG. 70;

FIG. 73 shows a side elevation view of the separation container of FIG.68 showing the plunger in a depressed position and the seal beingopened;

FIG. 74 shows a cross-sectional view of the separation container of FIG.73 taken along Section A-A;

FIG. 75 shows a top plan view of the separation container of FIG. 73;

FIG. 76 shows a detail view of the pellet region and first distal end ofthe plunger of Detail B in FIG. 74;

FIG. 77-85 show various views of a plunger 115 according to someembodiments discussed herein, including a perspective view (FIG. 77);side views (FIGS. 78, 81); detail views (FIGS. 79-80, 83); a top view(FIG. 85); a bottom view (FIG. 84); and a downward cross-sectional view(FIG. 82);

FIGS. 86-89 show various views of the flexible sealing member of theseparation container of FIGS. 1, 14, 32, 50, and 68;

FIG. 90 shows an exploded view of a separation container and centrifugecup according to some embodiments discussed herein;

FIG. 91 shows a side elevation view of the body of the separationcontainer of FIG. 90;

FIG. 92 shows a cross-sectional view of the body of FIG. 91 taken alongSection A-A;

FIG. 93 shows a cross-sectional view of the body of FIG. 91 taken alongSection D-D;

FIG. 94 shows a detail view of the second end of the body and capthreads of Detail B of FIG. 92;

FIG. 95 shows a detail view of the pellet region of the body of Detail Aof FIG. 92;

FIG. 96 shows a perspective view of a body of a separation containeraccording to some embodiments discussed herein;

FIG. 97 shows a bottom plan view of the body of FIG. 96;

FIG. 98 shows a cross-sectional view of the body of FIG. 97 taken alongSection E-E;

FIG. 99 shows a side elevation view of the separation container andcentrifuge cup of FIG. 90;

FIG. 100 shows a cross-sectional view of the separation container andcentrifuge cup of FIG. 99 taken along Section A-A;

FIG. 101 shows a side elevation view of a body of a separation containeraccording to some embodiments discussed herein;

FIG. 102 shows a cross-sectional view of the body of FIG. 101 takenalong Section A-A;

FIG. 103 shows a detail view of the second end of the body of Detail Aof FIG. 102;

FIG. 104 shows a detail view of the first end and pellet region of thebody of Detail B of FIG. 102;

FIG. 105 shows a detail view of the wall of the body of Detail C of FIG.103;

FIG. 106 shows a perspective view of the body of FIG. 101;

FIG. 107 shows a bottom plan view of the body of FIG. 106;

FIG. 108 shows a cross-sectional view of the body of FIG. 107 takenalong Section C-C;

FIG. 109 shows a side elevation view of a body of a separation containeraccording to some embodiments discussed herein;

FIG. 110 shows a cross-sectional view of the body of FIG. 109 takenalong Section A-A;

FIG. 111 shows a detail view of the second end of the body of Detail Aof FIG. 110;

FIG. 112 shows a detail view of the first end and pellet region of thebody of Detail B of FIG. 110;

FIG. 113 shows a perspective view of the body of FIG. 109;

FIG. 114 shows a bottom plan view of the body of FIG. 113;

FIG. 115 shows a cross-sectional view of the body of FIG. 114 takenalong Section C-C;

FIG. 116 shows a side elevation view oft a body of a separationcontainer according to some embodiments discussed herein;

FIG. 117 shows a cross-sectional view of the body of FIG. 116 takenalong Section A-A;

FIG. 118 shows a detail view of the second end of the body of Detail Aof FIG. 117;

FIG. 119 shows a detail view of the first end and pellet region of thebody of Detail B of FIG. 117;

FIG. 120 shows a perspective view of a body of a separation containeraccording to some embodiments discussed herein;

FIG. 121 shows a bottom plan view of the body of FIG. 120;

FIG. 122 shows a cross-sectional view of the body of FIG. 121 takenalong Section C-C;

FIG. 123 shows a side elevation view of a body of a separation containeraccording to some embodiments discussed herein;

FIG. 124 shows another side elevation view of the body of FIG. 124;

FIG. 125 shows a cross-sectional view of the body of FIG. 124 takenalong Section A-A;

FIG. 126 shows a detail view of the second end of the body of Detail Aof FIG. 125;

FIG. 127 shows a detail view of the first end and pellet region of thebody of Detail B of FIG. 125;

FIG. 128 shows a perspective view of the body of FIG. 123;

FIG. 129 shows a bottom plan view of the body of FIG. 123;

FIG. 130 shows a side elevation view of a body of a separation containeraccording to some embodiments discussed herein;

FIG. 131 shows another side elevation view of the body of FIG. 130;

FIG. 132 shows a cross-sectional view of the body of FIG. 131 takenalong Section A-A;

FIG. 133 shows a detail view of the first end and pellet region of thebody of Detail A of FIG. 132;

FIG. 134 shows a detail view of the second end of the body of Detail Bof FIG. 132;

FIG. 135 shows a perspective view of the body of FIG. 130;

FIG. 136 shows a bottom plan view of the body of FIG. 130;

FIG. 137 shows a side elevation view of a body of a separation containeraccording to some embodiments discussed herein;

FIG. 138 shows a cross-sectional view of the body of FIG. 137 takenalong Section A-A;

FIG. 139 shows a perspective view of the body of FIG. 137;

FIG. 140 shows a detail view of the second end of the body of Detail Aof FIG. 138;

FIG. 141 shows a detail view of the first end and pellet region of thebody of Detail B of FIG. 138;

FIG. 142 shows a perspective view of a body of a separation containeraccording to some embodiments discussed herein;

FIG. 143 shows a side elevation view of the body of FIG. 142;

FIG. 144 shows a cross-sectional view of the body of FIG. 143 takenalong Section A-A;

FIG. 145 shows a cross-sectional view of the body of FIG. 143 takenalong Section B-B;

FIG. 146 shows a bottom plan view of the body of FIG. 142

FIG. 147 shows a detail view of the first end and pellet region of thebody of Detail A of FIG. 144;

FIG. 148 shows a detail view of the second end of the body of Detail Bof FIG. 144;

FIG. 149 shows a side elevation view of the body of FIG. 142 having apull tab according to some embodiments discussed herein;

FIG. 150 shows a cross-sectional view of the body of FIG. 149 takenalong Section A-A;

FIG. 151 shows a perspective view of the body of FIG. 149;

FIG. 152 shows a bottom plan view of the body of FIG. 149;

FIG. 153 shows a detail view of the pull tab, first end of the body, andpellet region of Detail C of FIG. 150;

FIGS. 154-157 show various views of a sample collecting vessel accordingto some of the embodiments discussed herein;

FIG. 158 shows MALDI-TOF ID Results for Recovered Suspensions in Example1;

FIGS. 159-160 shows a comparison of historical colony control resultsversus VITEK2 AST card results for the Suspension of Example 1;

FIG. 161 shows a side elevation view of another embodiment of aseparation container having a raised plunger according to someembodiments discussed herein;

FIG. 162 shows a top plan view of the separation container of FIG. 161;

FIG. 163 shows a cross-sectional view of the separation container ofFIG. 161;

FIG. 164 shows another side elevation view of the separation containerof FIG. 161;

FIG. 165 shows another cross-sectional view of the separation containerof FIG. 161;

FIG. 166 shows a portion of the cross-sectional view of FIG. 165 showingDetail B;

FIG. 167 shows another side elevation view of the separation containerof FIG. 161;

FIG. 168 shows another top plan view of the separation container of FIG.161;

FIG. 169 shows another side elevation view of the separation containerof FIG. 161;

FIG. 170 shows a cross-sectional view of the separation container ofFIG. 169 taken along section B-B with the plunger in a piercing positionaccording to some embodiments discussed herein;

FIG. 171 shows a portion of the cross-sectional view of FIG. 170 showingDetail C;

FIG. 172 shows another side elevation view of the separation containerof FIG. 161 with the plunger down according to some embodimentsdiscussed herein;

FIG. 173 shows a top elevation view of the separation container of FIG.172;

FIG. 174 shows a cross-sectional view of the separation container ofFIG. 172 taken along section A-A;

FIG. 175 shows another side elevation view of the separation containerof FIG. 172;

FIG. 176 shows a cross-sectional view of the separation container ofFIG. 175 taken along section B-B;

FIG. 177 shows a portion of the cross-sectional view of FIG. 176 showingDetail A;

FIG. 178 shows an exploded view of the separation container of FIG. 161;

FIG. 179 shows another exploded view of the separation container of FIG.161;

FIG. 180 shows an exploded view of another embodiment of a separationcontainer according to some embodiments discussed herein;

FIG. 181 shows a portion of the exploded view of FIG. 180;

FIGS. 182-185 and 187-193 show a plunger and a retainer according tosome embodiments discussed herein;

FIG. 186 shows the plunger and retainer of FIGS. 182-185 and 187-193inserted into a body according to some embodiments discussed herein;

FIG. 194 shows a separation container having a sample collecting vesselaccording to some embodiments discussed herein;

FIG. 195 shows a cross-sectional view of the separation container ofFIG. 194;

FIG. 196 shows a perspective view of the separation container of FIG.194;

FIG. 197 shows a portion of the cross-sectional view of FIG. 195 showingDetail F;

FIG. 198 shows an exploded view of a two-piece coupling cap according tosome embodiments discussed herein;

FIG. 199 shows a side elevation view of the two-piece coupling cap ofFIG. 198;

FIG. 200 shows a cross-sectional view of the two-piece coupling cap ofFIG. 198;

FIG. 201 shows a side elevation view of another embodiment of a body andcoupling cap according to some embodiments discussed herein;

FIG. 202 shows a cross-sectional view of the body and coupling cap ofFIG. 201;

FIG. 203 shows a portion of the cross-sectional view of FIG. 202 showingDetail A;

FIG. 204 shows a portion of the side view of FIG. 205 showing Detail B;

FIG. 205 shows another side view of the body and coupling cap of FIG.201;

FIG. 206 shows a side elevation view of another embodiment of a body andcoupling cap according to some embodiments discussed herein;

FIG. 207 shows a cross-sectional view of the body and coupling cap ofFIG. 206;

FIG. 208 shows a portion of the cross-sectional view of FIG. 207 showingDetail A;

FIG. 209 shows another side view of the body and coupling cap of FIG.206;

FIG. 210 shows a portion of the side elevation view of the body andcoupling cap of FIG. 209 showing Detail B;

FIG. 211 shows an exploded view of the body and coupling cap of FIG.206;

FIG. 212 shows a side elevation view of yet another embodiment of aseparation container according to some embodiments discussed herein;

FIG. 213 shows a cross-sectional view of the separation container ofFIG. 212;

FIG. 214 shows a portion of the cross-sectional view of the separationcontainer of FIG. 212 showing Detail A;

FIG. 215 shows another side elevation view of the separation containerof FIG. 212;

FIG. 216 shows a cross-sectional view of the separation container ofFIG. 215;

FIG. 217 shows a portion of the cross-sectional view of FIG. 216 showingDetail B;

FIG. 218 shows a side elevation of the separation container of FIG. 212with the plunger down according to some embodiments discussed herein;

FIG. 219 shows a cross-sectional view of the separation container ofFIG. 218 showing the rheological control member having floated to thetop of the body according to some embodiments discussed herein;

FIG. 220 shows an exploded view of the separation container of FIG. 212;

FIG. 221 shows a side elevation view of the plunger, retainer, andrheological control member of FIG. 213;

FIG. 222 shows a cross-sectional view of the plunger, retainer, andrheological control member of FIG. 221;

FIG. 223 shows a finite element analysis of the deformation of a lowdensity polyethylene body during centrifugation;

FIG. 224 shows an exploded perspective view of another separationcontainer according to some embodiments discussed herein;

FIG. 225 shows a side elevation view of the separation container of FIG.224;

FIG. 226 shows a cross-sectional view of the separation container ofFIG. 225 taken along line A-A;

FIG. 227 shows a top plan view of the separation container of FIG. 224;

FIG. 228 shows a side elevation view of the separation container of FIG.225 rotated ninety degrees;

FIG. 229 shows a cross-sectional view of the separation container ofFIG. 228 taken along line B-B;

FIG. 230 shows a partial cross-sectional view of a portion of theseparation container indicated as Detail B in FIG. 229;

FIG. 231 shows a top plan view of the separation container of FIG. 224;

FIG. 232 shows a side elevation view of the separation container of FIG.224 without a cap according to some embodiments discussed herein;

FIG. 233 shows a side elevation view of the separation container of FIG.232 rotated ninety degrees;

FIG. 234 shows a cross-sectional view of the separation container ofFIG. 233 taken along line B-B;

FIG. 235 shows a partial cross-sectional view of a portion of theseparation container indicated as Detail C in FIG. 234;

FIG. 236 shows a side elevation view of the separation container of FIG.224 without a cap and with the plunger depressed according to someembodiments discussed herein;

FIG. 237 shows a side elevation view of the separation container of FIG.236 rotated ninety degrees;

FIG. 238 shows a cross-sectional view of the separation container ofFIG. 237 taken along line B-B;

FIG. 239 shows a partial cross-sectional view of a portion of theseparation container indicated as Detail A in FIG. 238;

FIG. 240 shows a side elevation view of the body and coupling member ofthe separation container of FIG. 224 according to some embodimentsdiscussed herein;

FIG. 241 shows an exploded view of the body and coupling member of FIG.240;

FIG. 242 shows a partial side elevation view of a portion of the bodyand the coupling member indicated as Detail A in FIG. 240;

FIG. 243 shows a perspective view of the body of FIG. 224 according tosome embodiments discussed herein;

FIG. 244 shows a top plan view of the body of FIG. 243;

FIG. 245 shows a side elevation view of the body of FIG. 243;

FIG. 246 shows a cross-sectional view of the body of FIG. 245 takenalong line A-A;

FIG. 247 shows a bottom plan view of the body of FIG. 243;

FIG. 248 shows a partial cross-sectional view of a portion of the bodyindicated as Detail A in FIG. 246;

FIG. 249 shows a partial cross-sectional view of a portion of the bodyindicated as Detail B in FIG. 246;

FIG. 250 shows a partial side elevation view of a portion of the bodyindicated as Detail C in FIG. 245;

FIG. 251 shows a partial top plan view of a portion of the bodyindicated as Detail D in FIG. 244;

FIG. 252 shows a cross-sectional view of the body of FIG. 245 takenalong line C-C;

FIG. 253 shows a cross-sectional view of the body of FIG. 245 takenalong line D-D;

FIG. 254 shows a perspective view of the coupling member of FIG. 240according to some embodiments discussed herein;

FIG. 255 shows a side elevation view of the coupling member of FIG. 254;

FIG. 256 shows a cross-sectional view of the coupling member of FIG. 255taken along a plane that vertically bisects the coupling member;

FIG. 257 shows a bottom plan view of the coupling member of FIG. 240;

FIG. 258 shows a partial side view of a portion of the coupling memberindicated as Detail A in FIG. 255;

FIG. 259 shows a side elevation view of a plunger of the separationcontainer of FIG. 224 according to some embodiments discussed herein;

FIG. 260 shows a partial side elevation view of a portion of the plungerindicated as Detail A in FIG. 259;

FIG. 261 shows a perspective view of a plunger seal of the plunger ofFIG. 259;

FIG. 262 shows a perspective view of the plunger of FIG. 259 without theplunger seal according to some embodiments discussed herein;

FIG. 263 shows a bottom plan view of the plunger of FIG. 262;

FIG. 264 shows a top plan view of the plunger of FIG. 262;

FIG. 265 shows a side elevation view of the plunger of FIG. 262;

FIG. 266 shows a partial side elevation view of a portion of the plungerindicated as Detail A in FIG. 265;

FIG. 267 shows a side perspective view of the plunger of FIG. 265rotated ninety degrees;

FIG. 268 shows a partial side elevation view of a portion of the plungerindicated as Detail B in FIG. 265;

FIG. 269 shows a perspective view of a flexible sealing member of theseparation container of FIG. 224;

FIG. 270 shows a side elevation view of the flexible sealing member ofFIG. 269;

FIG. 271 shows a cross-sectional view of the flexible sealing member ofFIG. 270 taken along a plane that vertically bisects the flexiblesealing member;

FIG. 272 shows a top plan view of the flexible sealing member of FIG.269;

FIG. 273 shows a side elevation view of the flexible sealing member ofFIG. 269 being actuated;

FIG. 274 shows a cross-sectional view of the flexible sealing member ofFIG. 273;

FIG. 275 shows a side elevation view of the separation container ofFIGS. 224-239 having its flexible sealing member and plunger actuatedaccording to some embodiments discussed herein;

FIG. 276 shows a cross-sectional view of the separation container ofFIG. 275;

FIG. 277 shows an exploded perspective view of another separationcontainer with an end cap according to some embodiments discussedherein;

FIG. 278 shows a side elevation view of the separation container of FIG.277;

FIG. 279 shows a cross-sectional view of the separation container ofFIG. 277;

FIG. 280 shows a partial cross-sectional view of the separationcontainer indicated as Detail B in FIG. 279;

FIG. 281 shows a partial cross-sectional view of the separationcontainer indicated as Detail B in FIG. 280;

FIG. 282 shows a top plan view of the flexible sealing member of FIG.277;

FIG. 283 shows a side elevation view of the separation container of FIG.277 without an end cap according to some embodiments;

FIG. 284 shows a cross-sectional view of the separation container ofFIG. 283;

FIG. 285 shows a partial cross-sectional view of the separationcontainer indicated as Detail C in FIG. 284;

FIG. 286 shows a side elevation view of the separation container of FIG.277 having a plunger actuated according to some embodiments;

FIG. 287 shows a cross-sectional view of the separation container ofFIG. 286;

FIG. 288 shows a partial cross-sectional view of the separationcontainer indicated as Detail A in FIG. 287;

FIG. 289 shows a side elevation view of a flexible sealing memberaccording to some embodiments;

FIG. 290 shows a cross-sectional view of the flexible sealing member ofFIG. 289;

FIG. 291 shows a perspective view of the flexible sealing member of FIG.289;

FIG. 292 shows a top plan view of the flexible sealing member of FIG.289;

FIG. 293 shows a partial cross-sectional view of the separationcontainer indicated as Detail A in FIG. 290;

FIG. 294 shows a partial cross-sectional view of the separationcontainer indicated as Detail B in FIG. 290;

FIG. 295 shows a partial cross-sectional view of the separationcontainer indicated as Detail C in FIG. 290;

FIG. 296 shows a side elevation view of a plunger according to someembodiments;

FIG. 297 shows a perspective view of a plunger seal of the plunger ofFIG. 296 according to some embodiments;

FIG. 298 shows a partial side elevation view of the plunger indicated asDetail A in FIG. 296;

FIG. 299 shows another side elevation view of the plunger of FIG. 296;

FIG. 300 shows yet another side elevation view of the plunger of FIG.296;

FIG. 301 shows a perspective view of the plunger of FIG. 296;

FIG. 302 shows a partial side elevation view of the plunger indicated asDetail A in FIG. 299;

FIG. 303 shows a partial side elevation view of the plunger indicated asDetail B in FIG. 299;

FIG. 304 shows a partial side elevation view of the plunger indicated asDetail C in FIG. 299;

FIG. 305 shows a top plan view of the plunger of FIG. 296;

FIG. 306 shows a bottom plan view of the plunger of FIG. 296;

FIG. 307 shows a perspective view of an end cap according to someembodiments;

FIG. 308 shows another perspective view of the end cap of FIG. 307;

FIG. 309 shows a side elevation view of the end cap of FIG. 307;

FIG. 310 shows another side elevation view of the end cap of FIG. 307;

FIG. 311 shows a cross-sectional view of the end cap in FIG. 309 takenalong the line A-A;

FIG. 312 shows a cross-sectional view of the end cap in FIG. 310 takenalong the line B-B;

FIG. 313 shows a top plan view of the end cap of FIG. 307;

FIG. 314 shows a bottom plan of the end cap of FIG. 307;

FIG. 315 shows a cross-sectional view of the end cap in FIG. 310 takenalong the line C-C;

FIG. 316 shows a cross-sectional view of the end cap in FIG. 309 takenalong the line D-D;

FIG. 317 shows a side elevation view of a body of a separation containerwith a coupling member according to some embodiments;

FIG. 318 shows a partial side elevation view of the body and couplingmember of FIG. 317;

FIG. 319 shows an exploded perspective view of the body and couplingmember of FIG. 317;

FIG. 320 shows a perspective view of the body of FIG. 317 according tosome embodiments;

FIG. 321 shows a side elevation view of the body of FIG. 320;

FIG. 322 shows a cross-sectional view of the body of FIG. 321 takenalong line A-A;

FIG. 323 shows a partial cross-sectional view of the body indicated asDetail B in FIG. 322;

FIG. 324 shows a partial cross-sectional view of the body indicated asDetail A in FIG. 322;

FIG. 325 shows a partial cross-sectional view of the body indicated asDetail C in FIG. 321;

FIG. 326 shows a bottom plan view of the body of FIG. 320;

FIG. 327 shows a top plan view of the body of FIG. 320;

FIG. 328 shows a cross-sectional view of the body of FIG. 321 takenalong line D-D;

FIG. 329 shows a cross-sectional view of the body of FIG. 321 takenalong line C-C;

FIG. 330 shows a perspective view of the coupling member of FIG. 317according to some embodiments;

FIG. 331 shows a side elevation view of the coupling member of FIG. 330;

FIG. 332 shows a cross-sectional view of the coupling member of FIG.330;

FIG. 333 shows a partial side elevation view of the coupling memberindicated as Detail A in FIG. 331;

FIG. 334 shows a top elevation view of the coupling member of FIG. 330;

FIG. 335 shows a perspective view of a rheological control memberaccording to some embodiments;

FIG. 336 shows another perspective view of the rheological controlmember of FIG. 335;

FIG. 337 shows a bottom plan view of the rheological control member ofFIG. 335;

FIG. 338 shows a cross sectional view of the rheological control memberof FIG. 337 taken along line B-B;

FIG. 339 shows a perspective view of a retainer according to someembodiments;

FIG. 340 shows another perspective view of the retainer of FIG. 339;

FIG. 341 shows a side elevation view of the retainer of FIG. 339;

FIG. 342 shows a top plan view of the retainer of FIG. 339;

FIG. 343 shows a bottom plan view of the retainer of FIG. 339; and

FIG. 344 shows a cross-sectional view of the retainer of FIG. 342 takenalong line A-A.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter withreference to the accompanying drawings in which some but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Biological testing of microorganisms is a delicate, time-sensitive, andoften dangerous process that requires accuracy, precision, andpreferably speed. The testing and analysis is carefully controlled,particularly when working with pathogenic microorganisms, using arepeatable, robust process and apparatus that allows the operator toeasily, safely, sterilely, and quickly manipulate the sample.Accordingly, there is a technical problem of providing a controlled,repeatable process and apparatus that allows an operator to easily,safely, sterilely, and quickly prepare a sample for further testing.

Disclosed herein are methods and apparatuses for recoveringmicroorganisms from a test sample. Some methods of characterizing and/oridentifying microorganisms within a sample may require initiallyseparating (e.g., separating, isolating, or pelleting) themicroorganism, followed by recovery for subsequent downstream testing.The methods discussed herein may include separating, recovering,characterizing, and/or identifying a sample using a separationcontainer. The separation container may be configured to separate amicroorganism from a sample via centrifugation and to recover a portionof the sample for use or testing. The sample may include a liquidculture (e.g., a blood culture) from which the microorganism may beseparated. Embodiments of the present invention also allow whole bloodto be used as the sample without culturing beforehand. In some furtherembodiments, a culture medium may be used in a sample collecting vesselto culture any organisms present after the separating and recoverysteps. In some embodiments, the resulting separated microorganism may betested, either in its isolated form or resuspended in solution, in oneor more downstream tests, and the downstream testing may occur within asample collecting vessel attached to the body of the separationcontainer or may occur separately (e.g., the sample may be separatelydeposited onto a downstream testing apparatus).

As used herein, the term “pellet” is intended to encompass any sample ofmicroorganisms that has been compressed or deposited into a mass ofmicroorganisms. For example, microorganisms from a sample can becompressed or deposited into a mass at the bottom of a tube bycentrifugation. In one embodiment, the term includes a collection ofmicroorganisms (and/or components thereof) on the bottom and/or sides ofa container following centrifugation. In accordance with this invention,microorganisms may be pelleted away (e.g., as a substantially purifiedmicroorganism pellet) from non-microorganism components that mayotherwise interfere with characterization and/or identification.Non-microorganism components that are separated away from themicroorganisms may include non-microorganism cells (e.g., blood cells orother tissue cells, and/or their soluble fractions) and/or anycomponents thereof.

Prior separation devices and techniques suffer from many deficienciesthat hinder the testing process. For example, microorganisms may beseparated by lysing the sample and then repeatedly washing, decanting,and spinning the sample until the microorganism is substantiallyseparated. These processes often require multiple devices, additionaluser handling, and a high-degree of expertise and training, whileproducing less-than-optimal results. For example, when exposed tocentrifugal force, different species of microorganism may producevarying consistencies of pellet. Prior devices may fail to recover thepellet consistently depending on the consistency of the pellet. Further,substantial training is required to successfully recover a pelletwithout contaminating the sample, losing the sample, or exposing theuser to the microorganism. Moreover, prior separation and recoverytechniques were very harsh on the microorganisms, making it difficult toobtain the highly viable microbial cells needed for certain downstreamtests such as antibiotic susceptibility testing (AST), growth-basedidentification methods, or the culture of microorganisms recovered fromwhole blood. Prior separation devices also frequently damage themicroorganism such that downstream testing with viable samples isimprecise or impossible. The present separation container (e.g.,separation container 100) may allow an untrained user to recover apellet with minimal training and effort and with greater consistencythan prior devices.

The inventors have developed a separation container and associatedapparatus, systems, and methods that solve these deficiencies. Namely,the separation container and associated apparatus, systems, and methodsdescribed herein enable a user to separate a microorganism from a samplein fewer operations with only a single centrifugation step. Theseparation container and associated apparatus, systems, and methodsdescribed herein also enable a user to separate and test the samplewithout handling the microorganism and without destroying themicroorganism so that viable samples may be grown and tested downstream.

Test samples that may be separated (e.g., separated, isolated, orpelleted) using the separation containers of the present inventioninclude both clinical and non-clinical samples in which microorganismpresence and/or growth is, or may be suspected, as well as samples ofmaterials that are routinely or occasionally tested for the presence ofmicroorganisms. For example, the test sample can be the culture brothfrom a culture of a clinical or non-clinical specimen sample. In someembodiments, the test sample is a sample taken from a patient withoutfurther culturing, e.g., a whole blood sample, a urine sample, a nasalsample, a buccal swab sample, or the like. The present invention findsuse in both medical and veterinary applications. Typical specimensamples that may be cultured and subsequently subjected to a separationtechnique for separation, isolation, or pelleting of microorganismscontained therein, may include, blood, serum, plasma, platelets, redblood cells, white blood cells, blood fractions, joint fluid, urine,nasal samples, semen, saliva, feces, cerebrospinal fluid, gastriccontents, vaginal secretions, tissue homogenates, bone marrow aspirates,bone homogenates, sputum, aspirates, swabs and swab rinsates, other bodyfluids, and the like. Examples of non-clinical samples includefoodstuffs (e.g., milk, meat products, vegetables, fruits, beverages,and puddings), cell cultures, biopharmaceuticals, cosmetics, water,parenterally-administered fluids, and the like. The above specimen typesmay also be used as samples for the present invention without culturingbeforehand. As described further herein, the separation container mayproduce viable samples suitable for direct downstream testing withoutfurther culturing and without requiring washing steps.

In one embodiment, as described further herein, the separation device orcontainer may employ a density cushion (e.g., density cushion 101 shownin FIG. 3) for the separation of microorganisms from a test sample. Forexample, the density cushion may have a density that is less than themicroorganisms being separated but greater than the remainder of thesample. For example, a test sample known to contain or that may containmicroorganisms can be loaded over a density cushion contained within thedevice or container, and the container or device centrifuged to separate(e.g., separate, isolate, or pellet) the microorganisms from otherelements of the test sample. In accordance with this embodiment, theseparation device or container will have sufficient volume to hold adensity cushion and a test sample. In one embodiment, the container fitsor can be fitted into a centrifuge rotor.

The volume of the container can be from about 0.1 ml to about 50 ml,e.g., from about 0.5 ml to about 25 ml, from about 1 ml to about 15 ml,e.g., from about 1.5 ml to about 8 ml. If the separation is done on amicroscale, the volume of the container can be from about 2 μl to about100 μl, e.g., from about 5 μl to about 50 μl. In some embodiments usinga deformable or squeezable container, described below, the volume of thecontainer may be less than 2 ml. In some embodiments using a plunger,the volume of the container may be from 10 ml to 15 ml, from 10 ml to 50ml, from 15 ml to 50 ml, greater than or equal to 10 ml, or less than orequal to 50 ml.

In some embodiments, as discussed in more detail herein, the separationdevice or container can be preloaded with the density cushion. In someembodiments, a rheological control member (liquid or solid) can beplaced on top of the density cushion before the sample is laid orlayered on top in order to prevent any mixing of the density cushion andthe sample. For example, an annular barrier (e.g., rheological controlmember 200 described below) can be placed over the prepackaged densitycushion to prevent mixing of the density cushion with a test sampleadded at a later time. In yet another embodiment, the separation deviceor container can be preloaded with a density cushion and subsequentlypreloaded with a lysis solution. In some embodiments, the separationcontainer may be hermetically sealed to prevent contamination.

The separation of the microorganism may be carried out by acentrifugation step in which a test sample (e.g., a lysed sample) isplaced on top of the density cushion in the separation container and thecontainer centrifuged under conditions which allow the microorganisms tobe isolated (e.g., the microorganisms can form a pellet at the bottomand/or sides of the container). The separation container is centrifugedat a sufficient acceleration and for a sufficient time for themicroorganisms to pass through the density cushion and be separated(e.g., a pellet formed) from other components of the test sample. Thecentrifugation acceleration can be about 1,000×g to about 20,000×g,e.g., about 2,000×g to about 15,000×g, e.g., about 3,000×g to about10,000×g, etc. The centrifugation time can be about 30 seconds to about60 minutes, e.g., about 1 minute to about 30 minutes, e.g., about 2minute to about 10 minutes. The centrifugation can be carried out at atemperature of about 2° C. to about 45° C., e.g., about 15° C. to about40° C., e.g., about 20° C. to about 30° C. In accordance with thisembodiment, other components of the sample (e.g., non-microorganisms orcomponents thereof that may be present in the sample) stay on top of thedensity cushion or within the top portion of the density cushion. Thisseparation step isolates the microorganisms from the remaining materialsin the sample, such as plasma, culture media, cell debris, and/or othercomponents such as enzymes, sugars and nucleic acids that mightinterfere with testing of the recovered microorganisms. In oneembodiment, the density cushion also serves to separate livemicroorganisms from dead microorganisms (which do not pass through thedensity cushion). In another embodiment the density cushion does notcomprise a density gradient, either before or after the centrifugation.In other words, the separation container is not centrifuged for asufficient amount of time and/or acceleration for the material making upthe density cushion to form a density gradient.

First Embodiment

Turning to the figures, embodiments of the separation containerdescribed herein are shown. With reference to FIGS. 1-89, a firstembodiment of the separation container 100 is shown. As discussedherein, the entire separation container 100 or separable portionsthereof may be placed in a centrifugation assembly to separate amicroorganism from a sample disposed in the container. In someembodiments, the separation container 100 may include a body 105 inwhich the sample may be disposed and a plunger 110, 115 in the body forextracting the separated microorganism after centrifugation. Theseparation container 100 may further include a seal 120 for sealing afirst end 106 of the body 105 and holding the sample in the body 105until the user actuates the plunger 110, 115. The separation container100 may also include a flexible sealing member 125 and cap 130 assemblyclosing a second end 107 of the body 105 while allowing the user toactuate the plunger 110, 115. In some embodiments, the separationcontainer 100 may include a sample collecting vessel 135 into which theseparated microorganism may be extracted, and the vessel 135 may beattached to the body 105 via an adaptor 140 and threaded connector 145.After a pellet of microorganism is expressed into the sample collectingvessel 135, the vessel 135 may be vortexed to dissolve the pellet in amedia (e.g., saline) in the vessel. Further views of the samplecollecting vessel 135 are shown in FIGS. 154-157. In some furtherembodiments, the body 105 may include one or more brackets (e.g.,hexagonal bracket 150 shown in FIG. 1) for engaging the centrifugationassembly and providing support for the separation container. Thehexagonal bracket 150 may allow the user to grip the separationcontainer and may support the separation container in the centrifugeholder. In some embodiments, the hexagonal bracket 150 may adapt theseparation container to one or more different centrifuge holders, and insome embodiments, the hexagonal bracket 150 may suspend the base of theseparation container above the base of the centrifuge. The projectionsmay also aid in manufacturing the separation container. For example, theprojections may be an alignment aid. In some embodiments, theprojections are designed with one or more flat surfaces. For example,two opposing flat surfaces may be used instead of the hexagonalorientation. The one or more flat surfaces engage a socket that isconfigured to apply consistent torque when securing the seal (e.g., anut) to the bottom of the separation container.

With reference to FIGS. 1-13, the separation container 100 is shownhaving a body 105 with a plunger 110 disposed therein. The body 105includes a first end 106, which defines an opening in the body, and asecond end 107, which defines a second opening. The body 105 may includean internal chamber 108 that is sealed at the first end 106 with a seal120. The internal chamber 108 of the body 105 may be sealed at thesecond end 107 with a cap 130 and/or a flexible sealing member. In someembodiments, the body (e.g., bodies 105, 505, 705, 905) of theseparation container can be molded, blow-molded, or formed using otherwell-known techniques in the art. In general, any known plastic, glass,or transparent material, or the like, can be used for the separationdevice. In some embodiments, the body 105 may be made of a stiffmaterial, such as polypropylene or a flexible material such as lowdensity polyethylene. In embodiments using a plunger 110, 115 asdescribed herein, the body 105 may be rigid or substantially rigid.

The internal chamber 108 of the body 105 may define a diameter radial tolongitudinal axis 155 (shown in FIGS. 2, 8, and 11). In someembodiments, the diameter of the internal chamber 108 may narrow from acollection diameter at the second end 107 to a pellet diameter at thefirst end 106. In some embodiments, the collection diameter may bedefined in a collection region 102 of the internal chamber 108, thepellet diameter may be defined in a pellet region 104 of the internalchamber 108, and a tapered region 103 may connect the pellet region withthe collection region. In some embodiments, the diameter of the internalchamber 108 at the collection region 102 is greater than the diameter ofthe internal chamber 108 at the pellet region 104. The wall 109 of thebody 105 at the tapered region 103 can have an included angle of about20 to about 70 degrees, e.g., about 30 to about 60 degrees. In someembodiments, the included angle of the wall 109 of the body 105 at thetapered region 103 is preferably 40 degrees or less. In someembodiments, the angle between the wall 109 of the body 105 and alongitudinal axis of the body 105 at the tapered region is preferably 20degrees or less. In some embodiments, included angle of the wall 109 ofthe body 105 at the tapered region 103 is preferably from 10 degrees to40 degrees. In some embodiments, the angle between the wall 109 of thebody 105 and a longitudinal axis of the body 105 at the tapered regionis preferably 5 degrees to 20 degrees. In one embodiment, the lowernarrow portion is less than half of the total height of the container,e.g., less than about 40%, 30%, 20%, or 10% of the total height of thecontainer. The pellet region 104 may be a “capillary” section at thefirst end 106 of the body, and the pellet region 104 may be thenarrowest portion of the body 105 in which the microorganism isconfigured to collect during centrifugation.

In each of the embodiments discussed herein the seal 120 may be disposedacross the opening defined at the first end 106 of the body 105, and theseal 120 may be configured to seal the opening and prevent the samplefrom escaping prior to actuating the plunger 110, 115. In someembodiments, the seal 120 may be an openable membrane (e.g., as shown inthe embodiments of FIGS. 1-89) that may be punctured by the plunger 110,115 or torn off by other mechanical means. For example, the membrane mayinclude a foil seal or other puncturable membrane (e.g., foil, paper,wax, etc.) or a thin plastic wall molded with the rest of the body 105.In some embodiments, the membrane is a film, tape, or frangible sealconfigured to cover the opening at the first end 106 of the body 105.The membrane may be adhered or otherwise attached to the end surface(e.g., the annular surface surrounding the opening at the first end 106)of the body 105. In one embodiment, the first end 106 comprises atextured surface to strengthen the adherence of the seal 120 to annularsurface surround the opening at the first end 106. In some embodiments,the seal 120 may be a thin layer of molded material that is formed atthe first end 106 of the body 105 during molding of the body. The moldedmaterial may be integral with or formed secondarily to the body 105. Inone embodiment, force multipliers are associated with the seal 120,e.g., formed in the molded material, to direct the opening of the seal120 when punctured by the plunger 110, 115.

In some other embodiments, the seal may include a nut 510 and gasket 520(e.g., as shown in the embodiments of FIGS. 90-136). In some furtherembodiments, the seal may include a removable portion 720 that may bepermanently cut or removed (e.g., as shown in the embodiment of FIGS.137-144). In yet some other embodiments, the seal may include a moldedbreakaway section or pull tab 920 that is torn off by the user to openthe internal chamber 108 (e.g., as shown in the embodiments of FIGS.142-153).

The flexible sealing member 125 may be disposed at the second end 107 ofthe body 105 and may be configured to seal the opening in the body whilealso allowing a user to actuate the plunger 110 therethrough. Theflexible sealing member 125 may seal against the body 105, such as bycontacting the annular surface of the second end 107 surrounding theopening. With reference to FIGS. 86-89, detailed views of an exampleflexible sealing member 125 are shown in accordance with the embodimentsdiscussed herein. In some embodiments, the flexible sealing member 125may include an open end 126 and a closed end 127. The plunger (e.g.,plungers 110, 115 shown in FIGS. 1-22, 32-38, 49-85) may have a secondend (e.g., second distal end 114 shown in FIG. 3) inserted into the openend 126 of the flexible sealing member 125 to allow the user to depressthe flexible sealing member and actuate the plunger. In any embodimentdescribed herein, the plunger 110, 115 may further be attached oradhered to the flexible sealing member 125.

In some embodiments, the flexible sealing member 125 may define abellows gasket having a cylindrical body 129 with corrugated side walls(e.g., conertinaed wall segments). The depicted body 129 may terminateat a flange 128 which may be disposed across the second end 107 of thebody 105 (shown in FIG. 3) to seal the opening in the second end. Inoperation, the cylindrical body 129 of the flexible sealing member 125may crush by bending at the corrugations upon application of force bythe user on the closed end 127. During actuation, the closed end 127moving downwardly may cause the flexible sealing member 125 to press theplunger 110, 115 downwardly (e.g., as shown in FIGS. 8-13 and 73-76).

Turning back to FIGS. 1-13, the flexible sealing member 125 may be heldonto the body 105 using a cap 130. The cap 130 may include an opening131 through which a second portion of the flexible sealing member 125,including a portion of cylindrical body 129 and the closed end 127 mayextend. The cap 130 may then be threaded or otherwise attached to thebody 105 and may leave the closed end 127 of the flexible sealing member125 actuatable to the user. In such embodiments, the second distal end114 of the plunger 110 may also extend through the opening 131 whilebeing disposed within the cylindrical body 129 of the flexible sealingmember 125.

The separation container 100 may further include a sample collectingvessel 135 for receiving a discharged pellet for downstream testing. Inthis manner, the separation container 100 may collect and hold theseparated microorganism without requiring expression of the samplepellet into a different container, risking spillage or contamination.The sample collecting vessel 135 may be attached to the body 105 and maysurround the opening at the first end 106, such that the seal 120 may beinside the sample collecting vessel 135 and the vessel is configured tocollect any discharge from the internal chamber 108 through the firstend 106 of the body. As discussed below, the sample collecting vessel135 may be pre-loaded with saline or another diluent for preparing asuspension of the isolated sample.

With continued reference to FIGS. 1-13, an adaptor 140 may be connectedto the body 105, either by being adhered, welded, or otherwise fastenedthereto, or by being integrally molded therewith. The adaptor 140 mayengage a conical portion of the body 105. In some embodiments, theadaptor 140 may engage a threaded connector 145, either by beingadhered, welded, or otherwise fastened thereto, or by being integrallymolded therewith. The sample collecting vessel 135 in the depictedembodiment is threaded to the threaded connector 145 for securing thevessel to the body 105. With reference to FIGS. 27-31, detailed views ofan adaptor 140 are shown. The adaptor 140 may include a lower flange 146portion into which the threaded connector 145 (e.g., shown in FIG. 3)may be inserted. The adaptor 140 may further include a ridge 147 (e.g.,as shown in FIG. 31) within the lower flange 146 that is configured toengage the threaded connector 145. The v-shaped ridge 147 may define anenergy director for ultrasonic welding, which may allow for a betterseal between the ridge 147 and threaded connector 145. As discussedherein, the adaptor 140, body 105, and threaded connector 145 may bewelded (e.g., by induction, heat, or sonic welding), adhered, orotherwise fused or attached to one another, or they may be integrallymolded. In such embodiments, the end cap 1250 may be used to apply anupward force on the body 1205 during welding.

In some embodiments, the adaptor 140 and threaded connector 145 mayterminate above (e.g., vertically above in the orientation of FIGS. 2-3)the seal 120 and the first end 106 of the body 105. Said differently,the adaptor 140 and the threaded connector 145 may be disposed axiallybetween the first end 106 and the second end 107, which may allow thefirst end 106 and the seal 120 to rest on a surface of the centrifugeapparatus during centrifugation to prevent premature breach of the seal.In embodiments of the separation container shown herein withoutadaptors, one of ordinary skill in the art will appreciate that anadaptor according to any of the embodiments described herein may beattached or molded with any of the disclosed separation containers. Thebody 105 may further comprise the hexagonal bracket 150 for supportingthe separation container 100 in the centrifugation apparatus or duringdownstream (e.g., post-separation) testing.

Turning to FIGS. 14-26, the embodiment of FIGS. 1-13 is shown having arheological control member 200 defined about the longitudinal member 112of the plunger 110 and within the internal chamber 108 of the body 105.Unless otherwise described herein, the features of the embodiment ofFIGS. 14-26 may be identical to the embodiment of FIGS. 1-13. Therheological control member 200 may define a barrier 205 that partiallyrestricts flow through the internal chamber 108. The barrier 205 may bean annular structure disposed about the longitudinal member 112 of theplunger 110. With reference to FIGS. 23-26, detailed views of an examplebarrier 205 of the rheological control member 200 are shown. Inparticular, the barrier 205 may include an inner annular ring 206 and anouter annular ring 207 connected by one or more vanes 208 to restrictflow in the internal chamber 108 while still allowing some of the sampleto pass therethrough.

The rheological control member 200 may further include a filter 210 forrestricting the flow further. The filter 210 may seat in an annularspace 209 of the barrier 205 to form a single unit. The rheologicalcontrol member 200 may define an outer radius of the outer annular ring207 that is less than the radius of the collection region 102 of theinternal chamber 108 to allow the rheological control member to movefreely within the body 105. The rheological control member 200 mayfurther define an inner radius of the inner annular ring 206 that isgreater than a diameter of the plunger 110 above a shelf 175. Therheological control member 200 may thereby move freely with respect toboth the body 105 and the plunger 110.

In some embodiments, the rheological control member 200 may preventmixing of the sample with the density cushion, described herein, tomaintain the dual separating and purifying properties of the densitycushion. For example, when a user pours the sample into the collectionregion 102 and a liquid density cushion is present in the internalchamber 108, the sample may mix with the density cushion and impair theseparation of the microorganism from non-microbial components duringcentrifugation. The rheological control member 200 allows the sample tosettle on the density cushion with little to no mixing. An exampledensity cushion 101 is shown in FIG. 3, and one of ordinary skill in theart will appreciate that any embodiment disclosed herein may include thedensity cushion in the same manner as depicted in FIG. 3.

In some embodiments, the rheological control member 200 may be buoyantin water and/or in a density cushion material. This may enable therheological control member 200 to float upwards (e.g., away from theseal 120) during centrifugation. In some embodiments, the rheologicalcontrol member 200 may define a specific density of 0.95 or lessrelative to water. In some embodiments, the rheological control member200 may define a specific density of 0.95 or less relative to thedensity cushion material. In some embodiments, the rheological controlmember 200 may define a specific density of 0.90 or less relative towater. In some embodiments, the rheological control member 200 maydefine a specific density of 0.90 or less relative to the densitycushion material. In some embodiments, the rheological control member200 may define a specific density of 0.85 or less relative to water. Insome embodiments, the rheological control member 200 may define aspecific density of 0.85 or less relative to the density cushionmaterial. In some further embodiments, the rheological control member200 may be buoyant in a mixture of water and density cushion material.

Turning to FIGS. 32-49, the embodiment of FIGS. 1-13 is shown havinganother embodiment of the rheological control member 300. Unlessotherwise described herein, the features of the embodiment of FIGS.32-49 may be identical to the embodiment of FIGS. 1-13. Although notdepicted in FIGS. 32-49, the embodiment of FIGS. 32-49 may also includea seal 120, adaptor 140, threaded connector 145, and/or samplecollecting vessel 135 according to any of the embodiments describedherein.

With reference to FIGS. 45-49, detailed views of the rheological controlmember 300 are shown. The rheological control member 300 may define abarrier 305 that partially restricts flow through the internal chamber108. The barrier 305 may be an annular structure disposed about thelongitudinal member 112 of the plunger 110. In particular, the barrier305 may include an inner annular ring 306 (shown in FIG. 49) and anouter annular ring 307 (shown in FIG. 49) connected by one or more vanes308 to restrict flow in the internal chamber 108 while still allowingsome of the sample to pass therethrough during centrifugation. In someembodiments, the inner annular ring 306 may seal against the plunger110, and the outer annular ring 307 may seal against the wall 109 of thebody 105 such that fluid must flow between the rings.

The rheological control member 300 may further include a frustoconicalsection 310 configured to snap a flange 311 into the barrier 305 andengage the inner annular ring 306 of the barrier. The frustoconicalsection 310 may further include a first annular seat 317 beneath theflange 311 for holding the barrier 305. In some embodiments thefrustoconical section 310 may include a conical body 313 that divertsfluid flow radially outward to further reduce mixing of the sample andthe density cushion. The rheological control member 300 may furtherinclude a lower annular member 315 that engages a second annular seat318 and a lower abutment 316 of the frustoconical section 310. The lowerannular member 315 may allow fluid to flow radially outward of itself,while reducing the turbulence of the sample flow.

In some embodiments, the frustoconical section 310 of the rheologicalcontrol member 300 may include an annular bore 312 extendingtherethrough. The bore 312 may receive the plunger 110, 115 therein, andmay allow the rheological control member 300 to slide relative to theplunger and may also form a seal between the rheological control memberand the plunger. With reference to FIGS. 45-47, the frustoconicalsection 310 may include an outwardly angled flange 314, such that fluidflow is configured to pass through the barrier 305, between the outerannular ring 307 and the inner annular ring 306, past the vanes 308, andthe fluid may then be deflected outwardly by the flange 314, lengtheningthe fluid flow path and reducing the risk of direct contact between thesample and the density cushion as the sample is loaded into the body.

In some embodiments, the frustoconical section 310 may be a flexiblevalve made of silicone or other elastomeric material. The frustoconicalsection 310 may be sealed against the barrier 305 during loading (e.g.,in the unstretched, normally-closed position of FIG. 47, fluid may notpass between the barrier 305 and the frustoconical section 310), and thebarrier 305 may be press fit within the separation container. Thisallows the separation container to be loaded without any concern ofdisturbing the density cushion. During centrifugation, however, thefrustoconical section 310 may stretch downwardly (e.g., to the openposition shown in FIG. 45) under the weight of the lower annular member315 during centrifugation to allow fluid to flow over the flange 314 andpast the frustoconical section 310. The lower annular member 315 may bea retainer positioned over the flexible frustoconical section 310, andthe lower annular member 315 may weigh the frustoconical section 310 sothat the frustoconical section distends during centrifugation. In thisconfiguration, the rheological control member 300 prevents fluidcommunication between the sample and the density cushion untilcentrifugation.

As detailed above, in some embodiments, the rheological control member300 may prevent mixing of the sample with the density cushion, describedherein, to maintain the filtering properties of the density cushion. Forexample, when a user pours the sample into the collection region 102 anda liquid density cushion is present in the internal chamber 108, thesample may mix with the density cushion and prevent separation of themicroorganism during centrifugation. The rheological control member 300allows the sample to settle in the internal chamber 108 with little tono mixing. In the embodiment shown in FIGS. 45-49, the barrier 305,frustoconical section 310, and lower annular member 315 may combine toreduce mixing of the sample with the density cushion.

Turning to FIGS. 50-56, the embodiment of FIGS. 1-13 is shown havinganother embodiment of the rheological control member 400. Unlessotherwise described herein, the features of the embodiment of FIGS.32-49 may be identical to the embodiment of FIGS. 1-13. In theembodiment shown in FIGS. 50-56, the rheological control member 400includes a substantially annular ring that serves the rheologicalcontrol functions described herein. In the embodiments described herein,the rheological control member may be any flow restriction that preventsthe density cushion and sample from mixing, which would destroy theefficacy of the density cushion, while still allowing microorganisms topass thereby during centrifugation. For example, the rheological controlmember may also include a plurality of polypropylene beads disposed inthe internal chamber 108. Although not necessarily required in the handsof a skilled user, the rheological control member may improve therobustness and precision of the devices, systems, and methods describedherein.

In particular, in some embodiments, the rheological control member 400may prevent mixing of the sample with the density cushion, describedherein, to maintain the filtering properties of the density cushion. Forexample, when a user pours the sample into the collection region 102 anda liquid density cushion is present in the internal chamber 108, thesample may mix with the density cushion and prevent separation of themicroorganism during centrifugation. The rheological control membersdescribed herein (e.g., rheological control members 200, 300, 400) allowthe sample to settle on the density cushion with little to no mixing.

In some embodiments the rheological control member 400 may be coupledwith the plunger 110, for example, by producing an annular ring that hasan inner diameter less than the outer diameter of the plunger 110. Insuch embodiments, the ring may be buoyant and may assist with flotationof the plunger 110 during centrifugation. In some other embodiments, theannular ring may be loosely retained about the plunger 110 and freelymovable along the axis 155 (shown in FIG. 3) of the plunger 110 duringoperation.

In some embodiments, the rheological control member 400 may be buoyantin water and/or in a density cushion material. This may enable therheological control member 400 to float upwards (e.g., away from theseal 120) during centrifugation and may cause the rheological controlmember to sit atop the density cushion. In some embodiments, therheological control member 400 may define a specific density of 0.95 orless relative to water. In some embodiments, the rheological controlmember 400 may define a specific density of 0.95 or less relative to thedensity cushion material. In some embodiments, the rheological controlmember 400 may define a specific density of 0.90 or less relative towater. In some embodiments, the rheological control member 400 maydefine a specific density of 0.90 or less relative to the densitycushion material. In some embodiments, the rheological control member400 may define a specific density of 0.85 or less relative to water. Insome embodiments, the rheological control member 400 may define aspecific density of 0.85 or less relative to the density cushionmaterial. In some further embodiments, the rheological control member400 may be buoyant in a mixture of water and density cushion material.

With reference to FIGS. 163, 165, 170, 174, 176, 178-180, 195, 213-214,216-217, and 219-222, another rheological control member 1042 is shown.The depicted rheological control member 1042 may include a central bore1080 for receiving a plunger 1010 therethrough. An outer surface of therheological control member 1042 may be configured to engage the wall1009 of the body 1005 of the separation container 1000. Between thecentral bore 1080 and the outer surface, the rheological control member1042 may be a substantially solid body, through which fluid may notpass.

In operation, the rheological control member 1042 may be interferencefit within the body 1005 prior to centrifugation. Said differently, anoutermost diameter of the rheological control member 1042 may be greaterthan the diameter of the body 1005. The interference fit may preventfluid from passing around the exterior of the rheological control member1042, between the rheological control member and the wall 1009. In astatic state (e.g., when the separation container 1000 is not beingcentrifuged), the internal chamber 1011 of the body 1005 may define afirst diameter radial to an axis extending in the longitudinal directionof the plunger 1010. The outermost diameter of the rheological controlmember 1042 may be greater than the first diameter.

During centrifugation, the body 1005 of the separation container mayflex slightly (e.g., as shown in the displacement diagram of FIG. 223)in a radially outward direction under the pressure of the liquid insidethe separation container 1000, increasing the diameter of the internalchamber 1011 and wall 1009 of the body 1005 to a second diameter. Insuch embodiments, the body 1005 may be made of an at least partiallyflexible material (e.g., low density polyethylene). With reference toFIG. 223, the displacement visualization shows a 0.66 mm displacement inthe x (radial) direction. The finite element analysis in FIG. 223 is ofa low density polyethylene body 1005 having a 15 mL volume. The appliedpressure during the FEA was 3.2 MPa at the base of the body 1005. Thesecond diameter may be greater than the outermost diameter of therheological control member 1042 such that the deformation of the wall1009 may release the interference fit between the rheological controlmember 1042 and the wall 1009 and allow fluid to flow therebetween.

In some embodiments, the rheological control member 1042 may be buoyantin water and/or the density cushion (e.g., having the same buoyantproperties described herein with respect to some plungers 110, 115,and/or may include a hollow space in its interior). In such embodiments,the rheological control member 1042 may float towards the second distalend 1007 of the body 1005 and out of the way. In some furtherembodiments, the rheological control member 1042 may be retained in awidened region 1008 of the body 1005 near the second distal end 1007once the diameter of the body 1005 narrows sufficiently to prevent therheological from moving freely in its original location. In someembodiments, a retaining notch or other frictional mechanism may be usedto retain the rheological control member 1042 at or near the seconddistal end 1007. In some embodiments, the rheological control member1042 may be disposed adjacent to the retainer 1032 after centrifugation.

In some embodiments, an annular shoulder 1061 may be formed in the wall1009 to ensure that the rheological control member 1042 is retained at aparticular axial position in the body 1005. For example, the rheologicalcontrol member 1042 may be retained at a position that allows thecomplete density cushion to be filled below the rheological controlmember, in the quantities described in the various embodiments herein,while leaving space above the rheological control member 1042 for thesample to be poured to allow the most usable volume in the separationcontainer while preventing mixing of the density cushion and samplebefore centrifugation. The diameter of the internal chamber of the body,radial to an axis of the body that is collinear with the length of thelongitudinal member 1012 of the plunger 1010, may change across theannular shoulder 1061. In some embodiments, the annular shoulder 1061may include a narrow side and a wide side relative to the axialdirection, such that the diameter of the internal chamber of the body1005 narrows when travelling in a direction from the second distal end1007 to the first distal end. In some embodiments, the annular shoulder1061 may serve as an axial limitation on the movement of the rheologicalcontrol member 1042, such that the rheological control member 1042 canmove freely upward, but may not fill the space of the density cushion.

The rheological control member 1042 may additionally have an annularshoulder 1062 that further improves the interference fit at the axiallocation where the two shoulders 1061, 1062 meet (e.g., as shown in FIG.217). The outermost diameter of the rheological control member 1042 maybe defined at the wide side of the annular shoulder 1062.

The central bore 1080 of the rheological control member 1042 may be slipfit with the outer diameter of the plunger 1010 such that the plungermay move relative to the rheological control member. In someembodiments, as shown in FIGS. 213, 214, 216, 219, and 220-222, a gasket1043 may be provided that seals the gap between the rheological controlmember 1042 and the plunger 1010 before centrifugation. The gasket 1043may be a flexible, circumferential component that extends about theplunger 1010. A protrusion 1053 of the gasket 1043 may engage acorresponding circumferential groove 1054 to prevent slipping. In someembodiments, the gasket 1043 may include an angled engagement surface1051 oriented downward (e.g., axially toward the first distal end 1006)and radially outward from the plunger. The rheological control member1042 may have a corresponding angled engagement surface 1052 at itsdistal end. During operation, the weight of the sample on therheological control member 1042 may compress the angled engagementsurface 1052 of the rheological control member 1042 onto the angledengagement surface 1051 of the gasket 1043, which may compress thegasket and increase the strength of the seal between the two. Oncecentrifugation begins, the rheological control member 1042 may be liftedoff of the gasket 1043 towards the second distal end 1007 as describedabove.

Due to the limited or no fluid flow being permitted between the spaceabove the rheological control member 1042 and the space below therheological control member 1042 when the rheological control member 1042is in its centrifugation position (e.g., the position shown in FIG.213), the density cushion may not mix with the sample prior tocentrifugation. In some embodiments, the separation container 1000 maybe packaged, stored and/or shipped with the density cushion, rheologicalcontrol member 1042, and plunger 1010 in position without leaking. Insome embodiments, the separation container may be pre-spun prior toloading a sample to settle the density cushion in the internal chamber1011. As detailed above, the rheological control member 1042 may besubstituted for any of the rheological control members described herein,and may be fitted to any of the plungers 110, 115, 1010 describedherein. Similarly, the other rheological control members describedherein may be applied to the plunger 1010 shown in FIGS. 166-223.

Referring back to FIGS. 1-67, in some embodiments, the plunger 110 mayinclude a longitudinal member 112 extending along an axis 155 (shown inFIG. 2) of the body 105. The plunger 110 may have a point 113 at a firstdistal end of the longitudinal member 112 configured to puncture theseal 120. The plunger 110 may further have a second distal end 114 thatis actuatable by a user to puncture the seal 120 and express the pelletas discussed herein. In a centrifugation configuration (e.g., as shownin FIGS. 2-7), the seal 120 is intact, and the plunger 110 may be sealedwithin the body 105. The plunger 110 may be actuated by depressing theflexible sealing member 125, which contacts and depresses the seconddistal end 114 of the plunger, to break the seal 120 and express thepellet from the first end 106 of the body 105 (e.g., as shown in FIGS.8-13) as described herein.

With reference to FIGS. 57-67, detailed views of the plunger 110 of theembodiment of FIGS. 1-56 are shown. The plunger 110 may include thelongitudinal member 112, which generally extends from the point 113 atthe first distal end to the second distal end 114. The point 113 may bedefined on a blade 160 at the first distal end of the longitudinalmember 112. By comparison between FIGS. 59 and 67, the blade 160 maydefine a first, wider diameter in a first radial direction (e.g., radialto the longitudinal axis 155 shown in FIG. 2), and a second, narrowerdiameter (e.g., the width of the blade in FIG. 59) in a second radialdirection perpendicular to the first radial direction and the axis. Forexample, each of FIGS. 3, 9, 16, 34, 49, 52, 57, 58, and 59 illustratesan edge-on view of the blade 160 with the second, narrower diametershown, and each of FIGS. 6, 7, 12, 13, 19, 20, 22, 37, 38, 55, 56, 64,and 67 illustrates the wider diameter of the blade.

In some embodiments, the point 113 is shaped to rupture the seal 120efficiently when the plunger 110 is activated. For example, the point113 may be conical-shaped and have a range of bevel angles relative tothe elasticity of the seal 120. In some embodiments, the point 113 isknife-shaped, chisel-shaped, hypodermic needle-shaped, or has multipleplanes, e.g., an X-shaped cross section, or the like. In one embodiment,the point 113 is shaped to create a void around the edge of the openingwhen the seal is punctured. In this embodiment, the void reduces captureof microorganism between the punctured seal 120 and the body 105. Infurther embodiments, the point 113 is designed to puncture the seal 120but not to tear away parts of the seal 120 that would then contaminatethe recovered sample. For example, the point 113 may be shaped to mirroror enhance force multipliers in the seal 120 to puncture the seal 120without breaking away any part of the seal after rupture.

The plunger 110 may further include one or more ribs 165, 170 forengaging the wall 109 of the body 105 defining the internal chamber 108.In particular, the ribs 165, 170 may be configured to engage the pelletregion 104 (shown in FIG. 3) of the body 105 to facilitate expression ofthe pellet (e.g., the separated microorganism). At least one of the ribs(e.g., stabilizing rib 165) may only extend partially about thecircumference of the plunger 110 and in some embodiments, may bedisposed to either side of the first, wider diameter of the blade 160(e.g., as shown in FIGS. 7, 22, 38, and 56). At least one of the ribs(e.g., upper sealing rib 170) may be defined above the blade 160 and mayextend circumferentially around the longitudinal member 112 of theplunger 110. In such embodiments, the sealing rib 170 may be on theopposite side of the blade 160 from the point 113. This sealing rib 170may define a plunger diameter d, and the sealing rib may be a generallycircular sealing surface that seals uniformly about the plungerregardless of the angular position of the blade 160. The sealing rib 170may be configured to engage the wall 109 of the body 105 and seal aportion of the internal chamber 108 below the rib 170 from a portion ofthe internal chamber above the rib. In particular, the plunger diameterd of the sealing rib 170 may be interference fit to the pellet diameter(e.g., slightly greater than the diameter of the body 105 at the pelletregion 104), such that the plunger 110, via rib 170, engages the pelletregion 104 during actuation of the plunger. In this manner, the plunger110 may fluidically isolate the pellet region 104 from the taperedregion 103 and the collection region 102 when actuating the plunger.

For example, FIGS. 7, 22, 38, and 56 show detail views of the plunger110 at the first end 106 of the body 105. In each depiction, the sealingrib 170 is configured to engage the wall 109 of the body 105 where itnarrows to the pellet diameter (e.g., at the start of the pellet region104). In such embodiments, the distance between the sealing rib 170 andthe point 113 may be less than or equal to the length of the pelletregion 104, such that the pellet region is fluidically isolated from theremainder of the internal chamber 108 prior to opening the seal 120.

In some further embodiments, the plunger 110 may further include a shelf175 at which point the diameter of the plunger 110 increases tosubstantially greater than the pellet diameter and greater than theplunger diameter d. This shelf 175, shown in FIGS. 3, 6-7, 9, 12, 16,19, 20, 22, 34, 37, 38, 49, 52, 55-58, and 64, may engage the transitionbetween the tapered region 103 and the pellet region 104 of the body 105to retain the plunger 110 within the separation container 100 and limitthe downward stroke of the plunger to only the travel distance necessaryto express the separated microorganism from the body 105.

The axial distance from the tip of the point 117 to the shelf 175 of theplunger 110 may be greater than the axial length of the pellet region104 to enable the plunger 110 to open the seal 120 without fallingentirely out of the body 105 or contaminating the separated sample inthe sample collecting vessel 135. Moreover, the axial distance from thetip of the point 117 to the sealing rib 170 may be less than or equal tothe axial length of the pellet region 104 to enable the plunger 110 toseal the pellet region 104 from the remainder of the internal chamber108 prior to opening the seal 120. In addition, the distance between theshelf 175 and the sealing rib 170 may be less than the axial length ofthe pellet region 104, such that the seal between the sealing rib 170and the wall 109 is not broken when the plunger 115 reaches its stoppingpoint.

With reference to FIGS. 68-89, an embodiment of the separation container100 is shown having a second plunger 115 and having a threaded adaptor240 integral with the body 105. Unless otherwise described herein, theembodiment of FIGS. 68-89 is identical to the embodiment of FIGS. 1-13As shown in FIGS. 70, 72, 74, 76-79, 81, and 84, the plunger 115 mayinclude a tapered conical first distal end 118 having a conical point117 rather than a flat blade (e.g., blade 160 shown in the embodiment ofFIGS. 1-67). The plunger 115 may define a sealing rib 270 and shelf 275that operate in substantially the same way as the sealing rib 170 andshelf 175 of the plunger 110 of FIGS. 1-67 shown and described herein.In particular, the pellet region 104 of the body 105 may be defined by asubstantially cylindrical portion of the body 105 against which thesealing rib 270 may move during actuation of the plunger 115. Asdiscussed above, the pellet region 104 may be a “capillary” section atthe first end 106 of the body, and the pellet region 104 may be thenarrowest portion of the body 105.

In some embodiments, when the sealing rib 270 is engaged with thecylindrical wall 109 of the body 105 at the pellet region 104, theportion of the internal chamber 108 on the side of the first end 106 ofthe body 105 may be fluidically separated from the remaining portion ofthe internal chamber 108 by the sealing action of the sealing rib 270.With reference to FIG. 79, the sealing rib 270 may define the plungerdiameter d₂. As also discussed above, the plunger diameter d₂ may begreater than the pellet diameter of the pellet region 104. In oneembodiment, the plunger diameter may be interference fit within thecylindrical wall 109 at the pellet region to form the seal thereagainst.

As also shown above, the plunger 115 may include a shelf 275, whichconstrains the downward movement of the plunger 115 in the body 105. Forexample, with reference to FIG. 76, the shelf 275 may define a widerportion of the longitudinal member 116 of the plunger 115. Inparticular, the shelf 275 may define a diameter that is greater than thediameter of the body 105 in the pellet region and less than the diameterof the body in the collection region 102. In such embodiments, the shelf275 may engage the wall 109 of the body 105 at the tapered region 103proximate the start of the pellet region 104 to prevent the point 117 ofthe plunger 115 from continuing further out of the body 105.

As discussed above with respect to the plunger 110 shown in theembodiments of FIGS. 1-67, the axial distance from the tip of the point117 to the shelf 275 may be greater than the axial length of the pelletregion 104 to enable the plunger 115 to open the seal 120 withoutfalling entirely out of the body 105 or contaminating the separatedsample in the sample collecting vessel 135. As also discussed above, theaxial distance from the tip of the point 117 to the sealing rib 270 maybe less than or equal to the axial length of the pellet region 104 toenable the plunger 115 to seal the pellet region 104 from the remainderof the internal chamber 108 prior to opening the seal 120. Moreover, thedistance between the shelf 275 and the sealing rib 270 may be less thanthe axial length of the pellet region 104, such that the seal betweenthe sealing rib 270 and the wall 109 is not broken when the plunger 115reaches its stopping point.

With reference to FIGS. 1, 3, 6, 7, 9, 12, 13, 14, 16, 19-22, 32, 34,37, 38, 49, 50, 52, 55, 56, 57-68, 70, 72, 74, and 76-85, in someembodiments, the plunger 110, 115 may include one or more stabilizers180, 280 that align and center the longitudinal member 112, 116 of theplunger 110, 115 within the body 105. In some embodiments, the plunger110, 115 may include two or more stabilizers 180, 280. In someembodiments, the plunger 110, 115 may include three or more stabilizers180, 280. In some embodiments, the plunger 110, 115 may include four ormore stabilizers 180, 280. Two of the stabilizers 180, 280 in theaforementioned embodiments may be positioned opposite one anotherrelative to the longitudinal member 112, 116 of the plunger. In someembodiments, each stabilizer 180, 280 may extend perpendicular to theaxis 155 of the longitudinal member and perpendicular to the stabilizerson either side of each respective stabilizer. The stabilizers 180, 280may extend perpendicular to the axis 155 (e.g., as shown in FIGS. 2,69), and may extend from the longitudinal member 112, 116 to a positionproximate the body 105. The stabilizers 180, 280 may thereby include aclearance (e.g., a slip fit or greater clearance) between thestabilizers and the wall 109 of the body 105 to prevent misalignment ofthe plunger 110, 115 without hindering the plunger's actuation orpreventing fluid from flowing through the internal chamber 108. In someembodiments, the stabilizers 180, 280 may be positioned on thelongitudinal member 112, 116 at the axial position of the collectionregion 102, which may define the widest portion of the internal chamber108 relative to the axis 155.

In some further embodiments, the plunger 110, 115 may be buoyant inwater and/or in a density cushion material. This may enable the plunger110, 115 to float upwards (e.g., away from the seal 120) duringcentrifugation. In some embodiments, the plunger may define a specificdensity of 0.95 or less relative to water. In some embodiments, theplunger may define a specific density of 0.95 or less relative to thedensity cushion material. In some embodiments, the plunger may define aspecific density of 0.90 or less relative to water. In some embodiments,the plunger may define a specific density of 0.90 or less relative tothe density cushion material. In some embodiments, the plunger maydefine a specific density of 0.85 or less relative to water. In someembodiments, the plunger may define a specific density of 0.85 or lessrelative to the density cushion material. In some further embodiments,the plunger 110, 115 may be buoyant in a mixture of water and densitycushion material. In some embodiments, the plunger 110, 115 may besufficiently stiff to not overly deform when engaging the pellet region104.

In some embodiments, the depicted plungers need not be buoyant. Forexample, with reference to the plunger 1010 shown in FIGS. 161-223, theplunger 1010 may be supported by a retainer 1032 that is secured withinthe body 1005 of the separation container 1000. In the depictedembodiment, the retainer 1032 is press fit into the second distal end1007 of the body 1005 of the separation container 1000. The body 1005may additionally or alternatively include a widened region 1008 at thesecond distal end 1007 that prevents the retainer 1032 from slippingfurther into the body. In operation, the retainer 1032 may retain andsupport the plunger 1010 such that the plunger does not puncture theseal 1020 during centrifugation. For example, during centrifugation, thefluid in the separation container may place 350 psi of pressure on theseal 1020 when 3000 rcf (relative centrifugal force, 3000×gravitationalforce). In some embodiments, the body 1005 may include a bracket 1050for supporting the separation container as described with respect to thehexagonal bracket 150 herein.

The retainer 1032 and plunger 1010 may have a retention mechanism thatallows the plunger to be supported by the retainer during centrifugationbut also actuatable by a user after separation. With reference to FIGS.182-192, detailed illustrations of the retainer 1032 and plunger 1010are shown. In particular, the retainer 1032 includes one or moreretaining members 1033 that engage corresponding support member 1015 onthe plunger 1010. The support member 1015 may include one or morelocking projections 1016 that engage the respective retaining members1033 of the retainer 1032. In the depicted embodiment, the retainer 1032includes two retaining members 1033 and the plunger 1010 includes twolocking projections 1016 each separated from the other by 180 degreesabout the longitudinal axis of the plunger 1010.

With reference to FIGS. 184-185, the retaining members 1033 may includea support projection 1034 extending from the inner wall of the retainer1032 with a stop wall 1036 at one end of the support projection. Thesupport projection 1034 may further include one or more locking tabs1035. With reference to FIG. 193, the locking projections 1016 mayinclude a substantially C-shaped receiving area defined by a lower wall1017, a lateral wall 1018, an upper wall 1019, and a lip 1021. Thesefeatures may combine to receive and engage the corresponding retainingmember 1033 of the retainer 1032. For example, the support projection1034 (shown in FIGS. 184-185) of the retaining member 1033 may bereceived in the locking projection 1016 (shown in FIG. 193) with anupper surface of the lower wall 1017 engaging a lower surface of thesupport projection 1034. The locking tab 1035 (shown in FIGS. 184-185)and/or the lip 1021 may deflect when the retaining member 1033 isengaged with the locking projection 1016 such that the locking tab 1035may be disposed in a space between the lateral wall 1018, the upper wall1019, and the lip 1021 to retain the plunger 1010.

During operation, the plunger may be inserted into the central openingof the retainer 1032 with the locking projections 1016 out of rotationalalignment with the retaining members 1033. When the locking projections1016 and retaining members 1033 are axially aligned, the plunger 1010may be rotated about its longitudinal axis to cause the supportprojection 1034 of the retainer 1032 to engage the locking projections1016 of the support member 1015. When engaged, the support member 1015is held axially in place such that the plunger 1010 cannot move up ordown the separation container relative to the retainer 1032, and thestop wall 1036 of the retaining members 1033 prevents the plunger 1010from rotating in one of two rotational directions. Gaps between theplunger 1010 and retainer 1032 may allow sample material and/or densitycushion to be added while the plunger 1010 and retainer 1032 areengaged. The retainer 1032 may also radially center the longitudinalmember 1012 of the plunger 1010 within the body 1005 while the plungeris engaged with the retainer. The plunger 1010 may be subsequentlydisengaged by rotating the plunger about its longitudinal axis in anopposite rotational direction, and the plunger may be free to moveaxially through the retainer 1032 when the locking projections 1016 arenot aligned with the retaining members 1033.

The plunger 1010 may include a tab 1022 at its second distal end 1014.The tab 1022 may give the second distal end 1014 of the plunger 1010 anon-cylindrical shape, which shape allows the user to grasp and turn theplunger through the sealing member 1025. For example, a radial pressureapplied to the tab 1022 at an angular position between the widest andnarrowest points of the second distal end 1014 may cause rotation in theplunger 1010. Thus, during centrifugation, the plunger 1010 may besupported by the retainer 1032 after which the user may release theplunger 1010 to express the pellet as described herein. Although shownin the embodiment of FIGS. 161-223, the retainer 1032 and support member1015 may be applied to any of the plungers 110, 115, 1010 in any of theembodiments shown and described herein.

As described below, the plunger 1010 shown in FIGS. 161-223 may expressthe pellet in substantially the same manner as the plungers 110, 115described in connection with one or more other embodiments herein. Insome embodiments, the plunger may include one or more ribs 1065, 1070,and 1072 as described herein. In some further embodiments, the plungermay include two sealing ribs 1070, 1072 that are vertically separatedfrom one another and extend circumferentially around the plunger 1010 ata location where each rib 1070, 1072 contacts the wall 1009 about itscircumference to seal the area above the respective rib from the areabelow the respective rib. In embodiments having two or more sealing ribs1070, 1072, one of the sealing ribs (e.g., the most distal rib 1070) mayprotrude through the body 1005 and out the central aperture 1044 of thecoupling cap 1040 (if used) as shown in FIG. 177 to ensure that thepellet is completely expressed, while also ensuring that the densitycushion and other remaining sample above the uppermost sealing rib 1072is prevented from leaving the body 1005 and contaminating the samplecollecting vessel 1038.

In some embodiments, the separation container 100 may use a densitycushion to facilitate separation and purification of the microorganismfrom the sample under centrifugation. The separation container 100 mayeither be loaded with a density cushion or may come pre-packaged with adensity cushion in the internal chamber 108 of the body 105. Thevolume/height of the density cushion in the internal chamber 108 shouldbe sufficient to achieve separation of the microorganisms, which passthrough the density cushion and are physically separated from othersample components. The volume will depend on the size and shape of theseparation container. In general, a volume of about 0.1 to about 25 mlcan be used. In some embodiments, a volume of about 0.2 to about 3 mlcan be used. In some embodiments, a volume of about 0.2 ml to about 0.5ml can be used. If the separation is performed on a microscale, thevolume of the density cushion can be about 1 μl to about 100 μl, and insome embodiments, about 5 μl to about 50 μl. The volume of sample laidor layered on top of the density cushion should be sufficient to provideenough microorganisms to produce a pellet suitable for testing. Ingeneral, any volume that fits into the container can be used. Forexample, a volume of about 0.1 ml to about 50 ml can be used. In someembodiments, a volume of about 0.2 ml to about 15 ml can be used. Insome embodiments, a volume of about 0.2 ml to about 1.5 ml can be used.If the separation is performed on a microscale, the volume of sample canbe about 1 μl to about 100 μl, and in some embodiments about 5 μl toabout 50 μl. The available space in the container for sample will dependon the size and shape of the container. In some embodiments, anintermediate layer (liquid or solid) can be placed on top of the densitycushion before the sample is laid or layered on top in order to preventany mixing of the density cushion and the sample. In one embodiment, theintermediate layer can be polyethylene beads. In another embodiment, asmall air bubble can be positioned between the density cushion and thesample to prevent mixing. In a further embodiment, the density cushioncan be layered on top of a high density material (e.g., aperfluorocarbon fluid) such that the microorganisms pass through thedensity cushion during the separation and collect at the interfacebetween the density cushion and the high density material.

The density of the cushion is selected such that the microorganisms inthe sample pass through the cushion while other components of the sample(e.g., plasma, blood culture broth, enzymes, sugars, nucleic acids)remain on top of the cushion or do not pass all of the way through thedensity cushion. Said differently, the density cushion may have adensity that is less than the microorganisms being separated and greaterthan the remaining sample material. The density cushion may also beselected to separate live microorganisms (which pass through thecushion) from dead microorganisms (which do not pass through thecushion). Suitable densities will depend on the material used in thedensity cushion and on the sample to be separated. In one embodiment,the density of the cushion is in the range of about 1.025 to about 1.220g/ml, e.g., about 1.030 to about 1.070 g/ml, about 1.040 to about 1.060g/ml or any range between about 1.025 to about 1.220 g/ml. In anotherembodiment, the density of the cushion is about 1.025, 1.030, 1.035,1.040, 1.045, 1.050, 1.055, 1.060, 1.065, 1.070, 1.075, 1.080, 1.085,1.090, 1.095, 1.100, 1.105, 1.110, 1.115, 1.120, 1.130, 1.140, 1.150,1.160, 1.170, 1.180, 1.190, 1.200, 1.210, or 1.220 g/ml.

The material for the density cushion can be any material that has theappropriate density range for the methods of this invention. In oneembodiment, the material is colloidal silica. The colloidal silica maybe uncoated (e.g., Ludox® (W.R. Grace, CT)) or coated, e.g., with silane(e.g., PureSperm® (Nidacon Intl, Sweden) or Isolate® (Irvine Scientific,Santa Ana, Calif.)) or polyvinylpyrrolidone (e.g., Percoll™, Percoll™Plus (Sigma-Aldrich, St. Louis, Mo.)). In one embodiment, the colloidalsilica exhibiting the least interference with spectroscopicinterrogation is selected. The colloidal silica may be diluted in anysuitable medium to form the proper density, e.g., balanced saltsolutions, physiological saline, and/or 0.25 M sucrose. Suitabledensities can be obtained with colloidal silica at a concentration ofabout 15% to about 80% v/v, e.g., about 20% to about 65% v/v. Anothersuitable material for density cushions is an iodinated contrast agent,e.g., iohexol (Omnipaque™ NycoPrep™, or Nycodenz®) and iodixanol(Visipaque™ or OptiPrep™). Suitable densities can be obtained withiohexol or iodixanol at a concentration of about 10% to about 25% w/v,e.g., about 14% to about 18% w/v, for blood culture samples. Sucrose canbe used as a density cushion at a concentration of about 10% to about30% w/v e.g., about 15% to about 20% w/v, for blood culture samples.Other suitable materials that can be used to prepare the density cushioninclude low viscosity, high density oils, such as microscope immersionoil (e.g., Type DF; Cargille Labs, New York), mineral oil (e.g.,Drakeol® 5, Draketex 50, Peneteck®; Penreco Co., Pennsylvania), siliconeoil (polydimethylsiloxane), fluorosilicone oil, silicone gel,metrizoate-Ficoll® (LymphoPrep™), e.g., at a concentration of about 75%to about 100% for blood culture samples, diatrizoate-dextran(PolymorphoPrep™), e.g., at a concentration of about 25% to about 50%for blood culture samples, carboxymethyl cellulose, hydroxypropylmethylcellulose, polyethylene oxide (high molecular weight), Pluronic® F127,Pluronic® F68, mixtures of Pluronic® compounds, polyacrylic acid,cross-linked polyvinyl alcohol, cross-linked polyvinyl pyrrolidine, PEGmethyl ether methacrylate, pectin, agarose, xanthan, gellan, Phytagel®,sorbitol, Ficoll® (e.g., Ficoll® 400 at a concentration of about 10% toabout 15% for blood culture samples), glycerol, dextran (e.g., at aconcentration of about 10% to about 15% for blood culture samples),glycogen, cesium chloride (e.g., at a concentration of about 15% toabout 25% for blood culture samples), perfluorocarbon fluids (e.g.,perfluoro-n-octane), hydrofluorocarbon fluids (e.g., Vertrel XF), andthe like as are well known in the art. In one embodiment, the densitycushion is selected from one or more of colloidal silica, iodixanol,iohexol, cesium chloride, metrizoate-Ficoll®, diatrizoate-dextran,sucrose, Ficoll® 400, and/or dextran in any combination. The densitycushion can also be made up of a combination of materials, e.g., acombination of colloidal silica and oil. Certain combinations of theabove compounds may be beneficial for the separation and downstreamtesting steps of the present invention. For example, combinations ofcompounds with different UV-quenching properties, such as cesiumchloride and Iohexol.

During operation, the separation container 100 containing the densitycushion may be loaded with a sample comprising one or more microorganismfor separation. As discussed herein, the sample may be lysed prior toloading the sample into the body. To load the sample, a user may removethe cap 130 and flexible sealing member 125 and pour the sample into thesecond end 107 of the body along the plunger 110, 115.

During loading, the rheological control member 200, 300, 400, if any,may prevent mixing of the density cushion and the sample and allow thesample to settle on top of the density cushion. The separation container100 may then be centrifuged to separate the microorganism from thesample and concentrate the microorganism. In particular, the separationstep can be carried out to separate the microorganisms from othercomponents of the sample (e.g., non-microorganisms or componentsthereof) and to concentrate the microorganisms into a separated (e.g.,isolated or pelleted) sample that can be recovered for culture and/oridentification and characterization purposes. The separation orpelleting step does not have to be complete, i.e., it is not requiredthat 100% separation occur. All that is required is that the separationof the microorganisms from other components of the sample be sufficientto permit downstream testing of the microorganisms without substantialinterference from the other components. For example, the separation canresult in a microorganism pellet that is at least about 10, 20, 30, 40,50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% pure or higher.

The separation container 100 may be centrifuged without the samplecollecting vessel 135 attached to the threaded connector 145 or threadedadaptor 240 respectively. In such embodiments, the seal 120 may bedisposed against a flat internal bottom of a centrifuge cup (e.g.,similar to centrifuge cup 540 shown in FIG. 90) and held in place by theflat surface of the centrifuge cup.

In some embodiments, the seal 120 need not be disposed against acentrifuge cap. For example, with reference to FIGS. 166-223, anembodiment of the seal 1020 is shown with a coupling cap 1040, 1140connecting and holding the seal to the first distal end 1006 of theseparation container 1000. Additionally or alternatively, the seal 1020may be sonic welded, heat or thermally welded, induction welded, orotherwise fused to the first distal end 1006. In some embodiments, theseal 1020 may be made of the same material as the body 1005 (e.g.,polypropylene). The coupling cap 1040, 1140 and/or sonic welding mayprevent the weight of the sample in the separation container 1000 fromprematurely opening the seal 1020 during centrifugation.

With reference to FIGS. 161-177, 194-197, and 212-220, a coupling cap1040 may be disposed on the first distal end 1006 of the body 1005 ofthe separation container 1000 about the pellet region 1004. The couplingcap 1040 may secure the seal 1020 to the body 1005 via compressionbetween a lower flange 1041 (shown in FIGS. 166, 171, 177, 197, and 200)of the coupling cap 1040 and the first distal end 1006 of the body 1005.In such embodiments, the coupling cap 1040 may apply even pressure aboutthe perimeter of the seal 1020 to prevent premature opening. In someembodiments the coupling cap 1040 may be sonic welded or otherwise fusedto the body 1005.

The coupling cap 1040 may further include a central aperture 1044through which the plunger 1010 may express the pellet during operation.In some embodiments, the central aperture 1044 may define a diameterthat is greater than or equal to the diameter of the pellet region 1004.The coupling cap 1040 may further include a circumferential v-shapedridge 1047 on the flange 1041. The v-shaped ridge 1047 may focus thesonic energy during attachment to the body 1005 for a better bond. Inpractice, the v-shaped ridge 1047 may flatten during the bonding processbetween the coupling cap 1040 and the body 1005.

In some embodiments, with reference to FIGS. 194-197, the coupling cap1040 may include one or more projections 1046 (e.g., circumferentialrings) extending from an outer surface of the coupling cap 1040. Theprojections 1046 may engage the sample collecting vessel 1038 andfrictionally retain the sample collecting vessel 1038 to the remainderof the separation container 1000. The coupling cap 1040, including theprojections 1046, may define an outer diameter that is greater than aninternal diameter of the sample collecting vessel 1038 so that thecoupling cap 1040 and sample collecting vessel are held together by aninterference fit.

With reference to FIGS. 180-181 and 198-200, the coupling cap 1040 isshown being formed in two pieces. In some embodiments, the coupling cap1040 may include an outer sealing member 1049 that includes theprojections 1046 and an inner cap 1048 that includes the flange 1041 andthat engages the first distal end 1006 of the body 1005. The inner cap1048 may include one or more cap projections 1039 that engagecorresponding recesses in the outer sealing member 1049 to hold the twotogether. In some embodiments, the outer sealing member 1049 may be madeof a softer, more flexible material (e.g., an elastomeric material) thanthe inner cap 1048, such that the inner cap 1048 may fuse with the body1005 (e.g., the inner cap 1048 may be made of the same material as thebody 1005) while the outer sealing member deforms against the inner cap1048 and sample collecting vessel 1038 to provide a better seal. Theouter sealing member 1049 may be overmolded onto the inner cap 1048and/or the body 1005. The coupling cap 1040 may otherwise operate insubstantially the same manner as detailed above with respect to FIGS.161-177, 194-197, and 212-220.

Turning to FIGS. 201-205, an embodiment of the coupling cap 1140 isshown having an enlarged inner cap 1148. In the depicted embodiment, theinner cap 1148 includes a frustoconical portion 1146 that abuts thetapered region 1003 of the body 1005. The frustoconical portion 1146 mayinclude a flange 1143 for resting or supporting the separation container1000, and the frustoconical portion 1146 may include a gripping section1142 that may be a circumferential portion of the inner cap 1148 thatangles toward and engages the body 1005. The operation of the couplingcap 1140 may otherwise be the same as disclosed with respect to thecoupling cap 1040 above. Each of the coupling caps 1040, 1140 hereinabove may be applied in connection with any of the separation containersdetailed herein, and coupling caps 1040, 1140 may be interchanged withor added to the foil securing methods described in connection with anyof the embodiments disclosed herein.

In some embodiments, with reference to FIGS. 206-211, the seal 1020 maybe attached directly to the first distal end 1006 of the body 1005. Forexample, in some embodiments, the seal 1020 may be sonic welded to thefirst distal end 1006. Fusing the seal 1020 directly to the body 1005may simplify manufacturing and increase the contact area between theseal 1020 and the body 1005 since no contact area is required for acoupling cap 1040, 1140. In such embodiments, the seal 1020 may be madefrom, or coated with, the same material as the body 1005. With continuedreference to FIGS. 206-211, an outer sealing member 1049 may be usedwithout an inner cap to seal the pellet region 1004 against the samplecollecting vessel 1038. In such embodiments, the body 105 may includeone or more cap projections 1039 to secure the outer sealing member 1049in substantially the same manner described above with respect to theinner cap 1048.

The container 100 may be centrifuged with the second end 107 of the bodydisposed radially inward with respect to the rotational axis of thecentrifuge and the first end 106 disposed radially outward, such thatthe apparent centrifugal force causes heavier portions of the sample(e.g., the microorganism) to collect at the first end 106.

As the sample is centrifuged, the microorganism may pass through thedensity cushion and collect at the first end 106 of the body 105, firstaccumulating in the pellet region 104 and, in some embodiments,subsequently at least partially filling the tapered region 103 withmicroorganism. The wall 109 of the body 105 at the tapered region 103may direct the microorganism radially inward towards the pellet region104. The centrifugation process may separate the microorganism into aconcentrated pellet resting against the seal 120 at the first end 106.As the sample is being centrifuged, the plunger 110, 115 may float withan upward buoyant force towards the second end 107 of the body 105. Thebuoyant force may be provided by the density of the plunger relative tothe density of the sample and density cushion as detailed above. In someembodiments, the plunger 110, 115 may include one or more flotationattachments to assist with lifting the plunger. In any of theabove-described embodiments, the buoyancy of the plunger 110, 115 mayprevent the plunger from prematurely piercing the seal 120 and preventthe plunger from prematurely separating the pellet region from the restof the internal chamber 108 (e.g., preventing the sealing rib 170, 270from prematurely engaging the wall 109 to prevent fluid flow into thepellet region 104).

In some embodiments, after centrifugation is complete, a concentratedpellet of microorganism, having the purity described above, will bedisposed in at least the pellet region 104 of the body 105, and nowashing or further centrifuging steps may be required. The user mayremove the separation container 100 from the centrifuge and conductdownstream analysis of the microorganism through any of the methods andtechniques described herein.

In some embodiments, the pellet may be removed from the body 105 of theseparation container 100, by actuating the plunger 110, 115, and theplunger 110, 115 may extract the pellet without compromising the purityor viability of the microorganism. In particular, the plunger 110, 115may be depressed to express the separated pellet of microorganism fromthe body 105. The user may press downwardly on the flexible sealingmember 125 in which the second distal end 114, 119 of the plunger 110,115 is disposed to actuate the plunger axially downwardly towards thefirst end 106.

As the plunger 110, 115 is depressed, the plunger may first seal againstthe wall 109 of the pellet region 104 to prevent fluid communicationbetween the pellet and the remaining sample and density cushion above.For example, in the embodiments shown in FIGS. 1-89, the sealing rib170, 270 may be lowered into contact with the cylindrical wall 109 atthe pellet region 104, which thereby seals the fluid below the rib fromthe fluid above the rib as two separate sub-compartments.

Continued downward movement of the plunger 110, 115 after sealing maycause pressure to build within the pellet region 104 between the sealingsurface of the plunger (e.g., sealing rib 170, 270) and the seal 120 atthe first end 106 of the body 105. As pressure builds and the plunger110, 115 continues to move downwardly, the point 113, 117 of the plungercontacts and opens the seal 120 by piercing through the seal orseparating the seal from the body 105. In some embodiments, the plunger110, 115 may require ca. 2 lbf of force to express the pellet. Openingthe seal 120 allows the pellet to be expressed from the internal chamber108 under the pressure created by the plunger 110, 115 being sealedagainst the wall 109 of the body 105. This back pressure may ensure thatthe pellet is cleanly and efficiently driven from the body 105.

In some embodiments, the pressure generated by the plunger 110, 115 isoptimized by controlling the volume of the internal chamber 108, thedistance between the point 113 of the plunger 110, 115 and the seal 120,and the position of the sealing surface on the plunger (e.g., sealingrib 170, 270). For example, the aforementioned distance, and thus thestroke of the plunger 110, 115, may be from 5 to 15 mm from initialmovement of the plunger to final contact between the shelf 175, 275 andthe wall 109. In one embodiment, the pressure is optimized to expressthe microorganism from the opening at a pressure appropriate forcapturing the microorganism in a tube. For example, the pressure may beenough to ensure that a majority of the microorganism is expressed fromthe tip without remaining on the edges of the opening (e.g., withoutremaining on the punctured seal remnants). The pressure may also be lowenough that the microorganism is released in a controlled spray insteadof an explosion that has the potential to aerosolize the microorganism.In one embodiment, the proportions of the plunger, the internal chamber,and the sealing surface are configured to generate a pressure thatexpresses the microorganism and disperses the microorganism in a sprayinto a downstream sample collection device without causing spray outsideof the downstream sample collection device.

After the seal 120 is opened, a portion of the seal may continue torestrict or prevent flow out the opening in the first end 106 (e.g., theremaining seal 120 narrows and restricts the annular opening between theplunger 110, 115 and the body 105). In some embodiments, additional flowresistance provided by the portions of the seal 120 that remain intactmay add even further back pressure against the pellet. This resistancecauses the internal pressure within the pellet region 104 to increase,which may cause the microorganism pellet to be expelled from the pelletregion 104 cleanly and under pressure without leaving substantialamounts of microorganism in the body. The recovered microorganism mayalso be viable, which may be needed for AST and/or further culturing.Moreover, the back pressure provided by the plunger 110, 115 issubstantially consistent between operations, due to the known distancesof travel of the plunger after sealing the wall 109 of the body 105 butbefore opening the seal 102. Because the back pressure provided by theplunger 110, 115 and/or seal 120 may be substantially predictablebetween uses, the user may more readily predict the force and timing ofthe expression of the pellet and avoid dangerous spillage or splatter ofthe microorganism.

Viability of the expelled, recovered microorganisms is dependent upon avariety of parameters such as age of the positive culture, and thechemical makeup of the selective lysis buffers and density cushionreagents. These reagents and their methods of use can be tailored todeliver microbial suspensions of high viability and vitality, asrequired for sensitive growth-based technologies such as antibioticsusceptibility testing (AST), or may be formulated to deliver cellsuspensions of higher purity, but potentially compromised viability, ifrequired for other downstream technologies such as MALDI-TOF and somemolecular, immunological, and immunochemical methods. Viability of therecovered microbial suspensions may be determined using culture of knowndilutions of the recovered suspension on agar plates, a technique commonto those skilled in the art, or the like. In some embodiments, therecovered microorganism may be viable, and in some embodiments, therecovered microorganism may not be viable.

In some embodiments, after the pellet has been expressed from the body105 but before the sealing portion of the plunger 110, 115 (e.g., thesealing rib 170, 270 in the embodiments detailed above) breaks throughthe opening at the first end 106, the shelf 175, 275 may engage the wall109 of the body 105 to stop further downward movement of the plunger. Inembodiments where the seal of the plunger 110, 115 (e.g., the sealingrib 170, 270 in the embodiments detailed above) is not broken, theremaining contents of the internal chamber 108 may not empty into thesample collecting vessel 135 or other receiving apparatus. In thismanner, the pellet may be expressed without risk of contaminating thesample due to inadvertent leakage or improper operation. In addition,maintaining the pellet under pressure from the plunger 110, 115 createsa positive pressure system that ensures that any leakage will flow fromthe pellet region 104 into the remainder of the internal chamber 108.Thus, any flaw in the seal between the plunger 110, 115 and the wall 109during expression of the pellet may result in microorganism being lostback into the internal chamber 108 but not contamination of themicroorganism in the pellet region 104 for downstream testing.

Further avoiding the risk of contamination or exposure to themicroorganism, the sample collecting vessel 135 may surround the openingat the first end 106 of the body 105 to receive the pellet without anyrisk of exposure to the user. In some embodiments, the sample collectingvessel 135 may be threaded onto the body 105 (e.g., via adaptor140/threaded connection 145 or threaded adaptor 240). Upon actuation ofthe plunger 110, 115 and expression of the pellet, the pellet may becollected in the sample collecting vessel 135 for downstream testing,including in situ culture. In some embodiments, the sample collectingvessel 135 may be glass, transparent plastic, or any other materialsuitable for the downstream testing needs of the user. In oneembodiment, for example, the sample collecting vessel 135 may be made ofany optically transparent material for later optical densityinterrogation of the microorganism through the wall of the samplecollecting vessel 135. In some other embodiments, the pellet may beexpressed directly into any other microorganism containment or storagevessel or any other testing apparatus as would be understood by a personof ordinary skill in the art in light of the present disclosure.

In some embodiments, the pellet collected in the sample collectingvessel 135 may be analyzed and/or tested in its concentrated form. Insome other embodiments, the sample collecting vessel 135 may include adiluent (e.g., saline or microbiological culture medium) for dilutingand resuspending the microorganism for downstream analysis and/ortesting. The diluent may be pre-packaged in the sample collecting vessel135 or may be loaded into the sample collecting vessel by the user in apredefined ratio of diluent to microorganism. The volume of the pellet104 may be known and may be predictable between samples. In particular,the volume of the pellet may be substantially the same as the volume ofthe pellet region 104, minus the volume of the first distal end 118, 160of the plunger 110, 115 below the sealing portion (e.g., below thesealing rib 170, 270) and minus certain, predictable losses due toleakage back into the internal chamber 108 and microorganism remainingin the pellet region 104 after the pellet is expressed. Because thevolume of the pellet may be anticipated and the downstream testing isknown, it may be possible to prepare the quantity of diluent in advanceand prepackage the sample collecting vessels 135 specific to theseparation container 100 and downstream test used. For example,antibiotic susceptibility testing (AST) may require a relatively dilutesample compared to other tests, and a sample collecting vessel that ispaired with AST may be given a precise quantity of diluent to producethe desired concentration of microorganism for testing.

In some embodiments, the axial location at which the plunger 110, 115seals against the wall 109 of the body 105 is dependent on the axiallength of the pellet region 104 (e.g., the sealing ribs 170, 270 mayengage the wall 109 at the start of the pellet region 104). Thus, asdiscussed above, in some embodiments, the volume expressed from thepellet region 104 may also be known and fixed, and may be dependent onthe volume of the pellet region. In the aforementioned embodiments, itis preferable that sufficient sample be loaded into the separationcontainer 100 to fill the pellet region 104 with separatedmicroorganism. For example, if a portion of the density cushion is leftin the pellet region, below the sealing ribs 170, 270, aftercentrifugation, this additional fluid may be expressed with theconcentrated pellet of microorganism. Thus, for some embodiments, tobetter predict the concentration of the recovered microorganism,sufficient sample material containing sufficient quantities orconcentration of microorganism should be used with a given sizedseparation container 100 to result in a separated microorganism pelletthat is at least large enough to fill the pellet region 104.

Second Embodiment

Turning now to FIGS. 90-136, a second embodiment of the separationcontainer 500 is shown without a plunger assembly. The separationcontainer 500 may include a body 505 with a deformable wall 509 thatallows a user to squeeze the body to extract the microorganism aftercentrifugation. In such embodiments, the separation body 505 may be madeof a flexible plastic such as low density polyethylene (LDPE) or otherflexible materials. Similar to the body 105 described above inconnection with the embodiment of FIGS. 1-89, the body 505 shown inFIGS. 90-136 may include a first end 506 at which the body may includean opening for extracting the microorganism pellet. Similarly, the body505 may include a second end 507 at which the body may include anopening for receiving the sample for centrifugation. The body 505 maydefine a collection region 502 and a pellet region 504 with a taperedregion 503 connecting the two. The collection region 502 may define awider radial diameter with respect to an axis 555 than the pellet region504, and the wall 509 at the tapered region 503 may angle radiallyinward from the collection region 502 to the pellet region 504 to directthe microorganisms into the pellet region during centrifugation. Thebody 505 may further define one or more projections (e.g., roundprojection 550) that operate in substantially the same manner as thehexagonal bracket 150 described herein for facilitating centrifugation,and one or more support struts 560 for stabilization and strength.

In some embodiments (e.g., as shown in FIGS. 92, 95, 98, 100, 102, 104,108, 110, 112, 115, 117, 119, 122, 125, 129, 132, and 135) the wall 509may taper inwardly for some or all of the wall's axial length in thepellet region 504. Said differently, in some embodiments, the radialdiameter of the pellet region 504 may be smaller at the first end 506than at the transition between the tapered region 503 and the pelletregion 504. In some embodiments, the wall 509 may taper uniformly fromthe transition between the tapered region 503 and the pellet region 504to the first end 506, as shown in FIGS. 117, 119, 122, 125, 129, 132,and 135. In some embodiments, the diameter of the pellet region mayrapidly narrow proximate the first end 506, as shown in FIGS. 92, 95,98, 100, 102, 104, 108, 110, 112, 115. In yet some further embodiments,the rapidly narrowing section may include a less tapered sectionimmediately adjacent the first end 506 as shown in FIGS. 92, 95, 98,100, 110, 112, and 115. In each of the aforementioned embodiments, thewall 509 may taper gradually in the remaining portion of the pelletregion 504 or may define a constant radius in the remaining portion.

With reference to FIGS. 90 and 99-100, the body 505 may include threads512 or other attachment mechanisms on its outer surface. The threads 512may engage a nut 510 to cover and seal the opening to the pellet region504 at the first end 506 of the body 505. The nut 510 may include agasket 520 or other sealing surface in its interior to prevent leakagefrom the internal chamber 508 of the body 505. As shown in FIG. 100,threading the nut 510 onto the body 505 may compress the gasket 520 withthe first end 506 to form a tight seal. The gasket 520 may be an RTVsilicone gasket, an expanded PTFE gasket, or other sealing gasketmaterial.

The separation container 500 may be inserted into an open end of acentrifuge cup 540 and the nut 510 may rest within a cavity 541 of thecup and against a closed end of the cup. In particular, the cup 540 maydefine a bottom wall 543 and a side wall 544 for supporting andstabilizing the separation container 500. As shown in FIG. 100, the nut510 may rest on the bottom wall 543 of the centrifuge cup 540. Duringcentrifugation, the centrifuge cup 540 may hang from one or more pivots542 such that the first end 506 of the body 505 is rotated radiallyoutward and the second end 507 of the body 505 is rotated radiallyinward during centrifugation.

With continued reference to FIG. 100, a rheological control member,shown here as a sphere 600, may be used in the internal chamber 508 forthe same purpose as the above-described embodiments. In particular, thesphere 600 may prevent mixing of the density cushion and the sampleduring loading of the sample into the body 505. As with theabove-described embodiments, in some embodiments, the sphere 600 may bereplaced with an annular structure, a plurality of smaller spheres, orany other rheological control member. The sphere 600 may be buoyant inthe density cushion and sample in accordance with the other embodimentsof the rheological control member described herein.

During operation, the loading and centrifuging processes occur insubstantially the same manner as described in the above embodiments. Insome embodiments, the sample may be lysed and loaded into the internalchamber 508, while the rheological control member (e.g., sphere 600) mayprevent mixing of the density cushion and the sample. The densitycushion may include substantially the same properties as discussed abovein connection with the embodiment of FIGS. 1-89. The separationcontainer 500 may then be centrifuged in the cup 540 with the nut 510and gasket 520 sealing the opening in the first end 506. Duringcentrifugation, the heavier microorganisms may pass through the densitycushion and settle in the pellet region 504, in which the microorganismsmay form a microorganism pellet.

After centrifugation the separation container 500 may be removed fromthe cup 540, and the nut 510 and gasket 520 may be removed by the user.To express the pellet, the user may then squeeze the deformable wall 509of the body 505 to expel the pellet into a container or testingapparatus. In some embodiments, the user may squeeze the deformable wall509 at any point above the pellet region 504. In such embodiments, thewall 509 may be made of a flexible material capable of being deformed bya user's hand.

As in the embodiments described above, the body 905 may be configured toengage a sample collecting vessel 135 for receiving the pellet aftercentrifugation. For example, the body 905 shown in connection with theembodiment of FIGS. 142-153 may include an adaptor (e.g., adaptor 140and threaded connector 145 shown in FIG. 1 or threaded adaptor 240 shownin FIG. 68) for engaging the vessel 135, or the body 905 may otherwisebe structured to engage a collection device. One of ordinary skill inthe art will appreciate in light of this disclosure that the propertiesof the separation container 500 and associated methods and apparatus mayinclude substantially the same features and steps as the otherembodiments described herein unless noted otherwise.

Third Embodiment

Turning now to FIGS. 137-141, a third embodiment of the separationcontainer 700 is shown having substantially the same features as theseparation container 500 shown in connection with FIGS. 90-136 exceptthat the first distal end 706 of the body 705 is removable and does nototherwise include any fluid flow paths through the first distal end.

The sealed first distal end 706 of the body 705 may ensure that fluiddoes not leak from the body 705 during centrifugation, and the firstdistal end 706 may include a removable portion 720. The removableportion 720 may be removed, for example, by cutting the end off at theentrance to the pellet region 704, to then squeeze or otherwise expressthe pellet from the body 705. As with the above-described embodiments,the wall 709 at the pellet region 704 may taper inwardly at any or allpoints between the transition from the tapered region 703 to the pelletregion 704 and the first distal end 706.

As in the embodiments described above, the body 705 may be configured toengage a sample collecting vessel 135 for receiving the pellet aftercentrifugation. For example, the body 705 shown in connection with theembodiment of FIGS. 142-153 may include an adaptor (e.g., adaptor 140and threaded connector 145 shown in FIG. 1 or threaded adaptor 240 shownin FIG. 68) for engaging the vessel 135, or the body 705 may otherwisebe structured to engage a collection device.

Fourth Embodiment

Turning to FIGS. 142-153, a fourth embodiment of the separationcontainer 900 is shown having substantially the same features as theseparation containers 500, 700 shown in connection with FIGS. 90-141except that the first distal end 906 of the body 905 includes a pull tab920 (shown in FIGS. 149-153) and does not otherwise include any fluidflow paths through the first distal end.

The sealed first distal end 906 of the body 905 may ensure that fluiddoes not leak from the body 905 during centrifugation, and the pull tab920 may be torn off to expose the entrance to the pellet region 904 andto then allow the user to squeeze or otherwise express the pellet fromthe body 905. FIGS. 142-148 show an example of the separation container900 after the pull tab 920 has been removed. As with the above-describedembodiments, the wall 909 at the pellet region 904 may taper inwardly atany or all points between the transition from the tapered region 903 tothe pellet region 904 and the first distal end 906.

As in the embodiments described above, the body 905 may be configured toengage a sample collecting vessel 135 for receiving the pellet aftercentrifugation. For example, the body 905 shown in connection with theembodiment of FIGS. 142-153 may include an adaptor (e.g., adaptor 140and threaded connector 145 shown in FIG. 1 or threaded adaptor 240 shownin FIG. 68) for engaging the vessel 135, or the body 905 may otherwisebe structured to engage a collection device.

Fifth Embodiment

Turning to FIGS. 166-223, a fifth embodiment of the separation container1000 is shown having a body 1005 including a first distal end 1006 and asecond distal end 1007. The body 1005 may include a wall 1009 definingan internal chamber 1011 divided into several regions, including acollection region 1002 (shown in FIG. 165), a widened region 1008between the collection region and the second distal end 1007, a taperedregion 1003, and a pellet region 1004. The plunger 1010 may includedistal ends 1013, 1014 and a shelf 1075 for preventing the plunger fromfalling out of the pellet region. In some embodiments, the portions ofthe wall 1009 in the pellet region 1004 may be reinforced with one ormore ribs 1045 extending in a longitudinal, axial direction of the body1005 for structural support. Each of the areas and features of the body1005 may be formed and operate according to any of the embodimentsdescribed herein.

Unless otherwise stated, the separation container 1000 may operate inthe same manner, may have the same properties, and may be made with thematerials and configurations of any embodiment described herein. Theseparation container 1000 of the fifth embodiment depicts a non-buoyantplunger 1010 that is retained by a retainer 1032 as described above. Thedepicted separation container 1000 also includes a rheological controlmember 1042 and gasket 1043 as described herein. In addition, thedepicted separation container 1000 may include a coupling cap 1040, 1140for securing the seal 1020 (e.g., a foil or other sealing membrane) tothe body 1005, and for coupling the body 1005 with a sample collectingvessel 1038, which may operate in the same manner as the samplecollecting vessels 135, 1038 described herein.

During assembly, the separation container 1000 may be assembled in thefollowing ordered steps: (1) add the density cushion to the internalchamber 1011; (2) connect the plunger 1010 with a retainer 1032; (3)insert the rheological control member 1042 onto the plunger 1010; (4)slide the gasket 1043 onto the plunger 1010 beneath the rheologicalcontrol member 1042; and (5) press fit the retainer 1032 into the tube,while also forming the interference fit between the rheological controlmember 1042 and the body 1005. In some embodiments the seal 1020 may beattached to the body 1005 prior to adding the density cushion. Theinterference fit may be created by inserting rods into the gaps betweenthe retainer 1032 and the plunger 1010 to press the rheological controlmember 1042 downwardly.

During testing, the separation container 1000 may be operated in thefollowing ordered steps: (1) To add the lysed sample to the tube, thecap 1030 and flexible sealing member 1025 may be removed; (2) the samplemay then be transferred to the tube, with the rheological control member1042 preventing bulk mixing of the sample with the density cushion sinceit is positioned between the two fluids; (3) the separation containermay be optionally pre-spun before adding the sample to move any densitycushion that may have migrated to the top chamber during shipping orstorage. The rheological control member 1042 may be constructed for asmall clearance between it and the wall 1009 of the body 1005 and mayinclude an interference fit when not being centrifuged as discussedabove. The rheological control member 1042 may also be constructed sothat it does not jam into the tube by bottoming out on the taperedregion 1003 of the body 1005. The testing may further include: (4)during addition of the sample the rheological control member 1042 mayfloat if optionally not interference fit, while still providing mixingprotection. The rheological control member 1042 may also have a recessedring in the top portion, which can trap any settling components such asresin or other particulates. These particles may stay in the recessedarea as the layering aid floats up the tube either during sampleaddition or centrifugation. The testing may further include: (5) afterthe sample is added, the cap 1030 and flexible sealing member 1025 maybe placed back on the tube; and (6) the entire separation container 1000may be placed in the centrifuge and spun. In embodiments where therheological control member 1042 is sealed against the body 1005 andplunger 1010, the sample may be added directly to the internal chamber1011 with a lytic agent and the entire separation container 1000 can bevortexed to lyse the sample before the centrifugation step.

In operation, the process for expressing the pellet is the sameactuation and foil piercing process described herein with respect toeach other embodiment, and in embodiments using a retainer 1032, anadditional step of applying horizontal pressure to the tab 1022 of theplunger 1010 may be used to disconnect the plunger from the retainer.Unless otherwise stated, features having the same reference numeral,name, or purpose in the assembly may be interchanged with one another inany of the embodiments described herein.

Sixth Embodiment

With reference to FIGS. 224-276 a sixth embodiment of the separationcontainer 1200 is shown. With reference to FIG. 224, an exploded view ofthe separation container 1200 is shown depicting a body 1205 having aseal 1220 to close an opening in one end and a cap 1230 and flexiblesealing member 1225 at the other. The separation container 1200 mayinclude a plunger 1210 having a plunger seal 1290, a rheological controlmember 1242, and a retainer 1232 attached thereto for ensuring smoothoperation and reducing contamination of the sample pellet as describedbelow. The separation container 1200 may further engage an end cap 1250for supporting the body 1205 and seal 1220 during centrifugation, andthe separation container may include a coupling member 1240 for couplingand sealing the body to a sample collecting vessel (e.g., the samplecollecting vessels 135, 1038 described herein).

With reference to FIGS. 225-230, assembled views of the separationcontainer 1200 are shown. In some embodiments, the separation container1200 may engage the end cap 1250 to support the seal 1220 and body 1205during centrifugation to allow the separation container to be adaptableto many different centrifuge cups while also improving manufacturabilityand quality. The end cap 1250 may hold the seal 1220 in place duringinduction welding in instances in which the seal is induction welded tothe tube. If the seal 1220 is heat sealed, the seal may be attachedbefore the end cap 1250 is applied. In each of the embodiments discussedherein, the end cap 1250 may support the seal 1220 from distending andrupturing during centrifugation.

The end cap 1250 may include a tapered portion 1251, which may be solidor include one or more ribs 1252 with gaps therebetween. The end cap1250 may include a substantially flat distal end 1253 and may include aflat flange 1254 at an opposite end. The flat distal end 1253 may beconfigured to engage the flat or rounded bottoms of various types andmodels of centrifuge cup, and the distal end 1253 may be narrower thanthe flange 1254 at the opposite end with the tapered portion 1251narrowing the diameter of the end cap between the two ends. In someembodiments, the tapered portion 1251 may be configured to engage thewalls of conical-shaped centrifuge cups. Thus, the end cap 1250 may bestructured to engage different models and structures of centrifugewithout needing different adaptors or sacrificing stability.

In some embodiments, the body 1205 may include a flange 1255 that isconfigured to engage the flange 1254 of the end cap. In someembodiments, the flange 1255 may be disposed circumferentially about thetapered region 1203 of the body 1205 and oriented towards the flange1254 of the end cap 1250, as shown in the embodiment of FIGS. 225, 226,228-234, and 236-238. In some embodiments, the flanges 1254, 1255 may beremovably or semi-permanently attached to each other. For example, insome embodiments, a threaded connection (not shown), snap connection(not shown), or other removable engagement may be formed between thebody flange 1255 and the end cap flange 1254. In some embodiments, theflanges 1254, 1255 may be adhered, welded, molded, or otherwisesemi-permanently fused to each other, such that the bond between theflanges must be mechanically broken to remove the end cap 1250. In someembodiments, the end cap 1250 may be press fit over the coupling member1240, such that the end cap does not need to be welded to the body 1205.In each instance, the end cap 1250 may be optionally welded to the body1205.

In some embodiments, the end cap 1250 may include a hex-shaped recess1257 in the distal end 1253. The recess 1257 may be used to hold theseparation container 1200 (e.g., by standing the separation containerand end cap 1250 on a vertical post (not shown)) when not in use, whenfilling the container, or during centrifugation. In some embodiments, apost or other rigid object (not shown) may be inserted into the recess1257 to assist with removal of the end cap 1250 after centrifugation.For example, in embodiments in which the end cap 1250 has its flange1254 semi-permanently attached to the flange 1255 of the body, the postor other rigid object may be a corresponding hex shape, such that thepost or other rigid object may be inserted into the recess 1257 and maybe used to twist or torque the semi-permanent connection apart. Themechanical separation of the end cap 1250 with a tool (e.g., the post orother rigid object referenced above) may reduce agitation of the sampleand prevent inadvertent contamination.

In some embodiments, the end cap 1250 may further include an internalplatform 1256 against which the first distal end 1206 of the pelletregion 1204 may rest, either with the body 1205 touching the platform1256 or with the seal 1220 sandwiched therebetween. The platform 1256may support the seal and prevent inadvertent rupture before the user isready to express the final concentrated pellet, including duringmanufacturing, assembly, packaging, loading, and centrifugation. Inaddition, the platform 1256 of the end cap 1250 allows a full sized seal1220 to be attached to the entire surface area of the distal end of thepellet region 1204 providing for improved manufacturability and animproved seal between the seal 1220 and body 1205.

With reference to FIGS. 224, 226, 229, 230, 232-242, and 254-258, acoupling member 1240 may be disposed on the body 1205 at or near thepellet region 1204 to facilitate coupling of the separation container1200 to a sample collecting vessel (e.g., the sample collecting vessels135, 1038 detailed herein) and/or to provide a further seal within theend cap 1250 to prevent leakage of the contents of the body 1205. Thecoupling member 1240 may include one or more circumferential ridges 1241to provide a seal against the sample collecting vessel. The couplingmember 1240 may further include a first, distal sealing surface 1243 anda second, upper sealing surface 1244, which may be pointed (e.g., likeridges 1241) or flat. The sealing surfaces 1243, 1244 and the ridges1241 may progressively decrease in their diameter when travellingtowards the first distal end 1206 of the pellet region 1204. Forexample, the first sealing surface 1243 may be the narrowest sealingsurface of the coupling member 1240 and the second sealing surface 1244may be the widest sealing surface of the coupling member with each ridge1241 therebetween progressively increasing in diameter, which mayfacilitate insertion of the separation container into the samplecollecting vessel while providing a strong seal.

With reference to FIG. 230, in some embodiments, the second sealingsurface 1244 may be wider than the inner diameter of a sample collectingvessel (not shown) (e.g., the sample collecting vessels 135, 1038 shownherein) and the second sealing surface 1244 may include a chamfer 1245for engaging the upper edge of the sample collecting vessel. The chamfer1245 may either create a seal against the upper rim of the samplecollecting vessel or may compress the second sealing surface 1244 inwardto fit the second sealing surface within the sample collecting vessel,while reducing the force required for engagement. In some embodiments,any other connector or coupling may be used to engage the body 1205 witha sample collecting vessel. The other connector or coupling may beattached to either the body 1205 or the sample collecting vessel or maybe a separate device.

The end cap 1250 may include a narrow, first wall 1246 near or adjacentto the platform 1256 and may include a wider, second wall 1247 above thefirst wall opposite the platform. The first wall 1246 and the secondwall 1247 (the inner and/or outer surfaces) may be parallel to the wallof the pellet region 1204 or may be tapered inwardly slightly such thatthe distal end closest to the platform 1256 is narrower than theopposite end for either wall segment 1246, 1247. The first wall 1246 andsecond wall 1247 may define diameters that are less than or equal to thediameters of the respective first sealing surface 1243 and secondsealing surface 1244 at least at the same respective axial positionsabove the platform 1256.

With continued reference to FIG. 230, the body 1205 may include one ormore circumferential ribs 1248, 1249 that provide structural rigidity tothe connection between the body and the coupling member 1240. Forexample, in the embodiment shown in FIG. 230, the body includes twocircumferential ribs 1248, 1249, one disposed proximate the taperedregion 1203 of the body and one disposed proximate the first distal end1206 of the pellet region 1204. In the embodiment shown in FIG. 230, afirst circumferential rib 1248 is embedded in a corresponding recess inthe first sealing surface 1243 of the coupling member 1240. Similarly,in the embodiment shown in FIG. 230, a second circumferential rib 1249is embedded in a corresponding recess 1260, 1261 in the second sealingsurface 1244 of the coupling member 1240. The coupling member 1240 andthe body 1205 may be attached via overmolding, elastically stretchingthe coupling member 1240 over the body 1205, or fusing multiple piecesof the coupling member 1240 together around the body 1205. In someembodiments, the coupling member 1240 may be made of a compliantmaterial such as elastomer. The coupling member 1240 may be overmoldedonto the body 1205. In some embodiments, the coupling member 1240 may bemade from silicone or thermoplastic elastomers such as Medalist®MD-12140.

With reference to FIGS. 241, 243, 245, 248, 250, 252, 256, and 257, thebody 1205 may also include one or more vertical ribs 1262, and thecoupling member 1240 may include one or more corresponding recesses 1263for receiving and engaging the vertical ribs 1262. In some embodiments,the body 1205 may include a base flange 1264 at the distal end of thepellet region 1204 for retaining the coupling member 1240 with its uppersurface and engaging the seal 1220 with its lower surface (e.g., viasonic welding).

An end of the coupling member 1240 opposite the base flange 1264 mayfurther abut a portion of the outer surface of the tapered region 1203,such that the combined upward retention from the base flange and thedownward retention from the tapered region encourage the coupling member1240 to a stable position (e.g., the position shown in FIG. 242). Insome embodiments, the coupling member 1240 may be disposed between theflange 1255 on the body 1205 in the tapered region 1203 and the baseflange 1264 such that the coupling member 1240 fits within the end cap1250. The coupling member 1240 may include bi-directional (e.g.,perpendicular) recesses (e.g., recesses 1260, 1261, 1263) to engage thecorresponding bi-directional ribs (e.g., ribs 1248, 1249, 1262) of thebody and prevent both axial and rotational movement of the couplingmember.

As discussed in connection with other embodiments herein, the body 1205may include a first distal end 1206, a second distal end 1207, a taperedregion 1203, a pellet region 1204, a collection region 1202, a widenedregion 1208, and a wall 1209, each of which may operate and may bestructured as described in each embodiment herein. The separationcontainer 1200 may further include a retainer 1232 attached to the body1205 in the widened region 1208. In some embodiments, with reference toFIGS. 243, 244, 249, and 251, the widened region 1208 of the body 1205may include one or more slots 1265 which may receive and secure theretainer 1232 therein. The retainer 1232 may be otherwise structured andoperable as described above with respect to the retainer 1032 to securethe plunger 1210 in a predetermined vertical position (e.g., a positionin which the upper edge of the retainer 1232 is coplanar with the seconddistal end 1207 of the body 1205). For example, with reference to FIG.259, the plunger 1210 may include a support member 1215 having thefeatures and structure shown and described in connection with thesupport member 1015 of the plunger 1010, and the retainer 1232 mayinclude retaining members 1233 and corresponding features having thefeatures and structure shown and described in connection with theretainer 1032 of the plunger 1010.

With reference to FIGS. 259-268, the plunger 1210 is shown in accordancewith some embodiments discussed herein. The plunger 1210 may include alongitudinal member 1216 coming to a conical point 1217 at a firstdistal end 1218. The plunger may include a piercing section 1270defining a cylindrical portion of the longitudinal member 1216 whichterminates with a taper at the point 1217. The piercing section 1270 mayoperate in substantially the same manner as the blade 160 or point 117discussed herein, and the piercing section 1270 and point 1217 may benarrower than the longitudinal member 1216 or the remaining portions ofthe plunger 1210 to allow for collection of additional sample in thepellet region 1204. In this manner, the relative diameters and sizes ofthe components shown in the figures are intended to be accurate asexample embodiments (e.g., the piercing section 1270 is depictednarrower than the remainder of the longitudinal member 1216).

In some embodiments, the plunger 1210 may include at least one sealingrib 1272 formed circumferentially about the longitudinal member 1216,and may include a tapered region 1271 between the sealing rib 1272 andthe piercing section 1270. This sealing rib 1272 may define the plungerdiameter d, and the sealing rib may be a generally circular sealingsurface that seals uniformly about the plunger. The sealing rib 1272 maybe configured to engage the wall 1209 of the body 1205 and seal aportion of the internal chamber 1211 below the rib 1272 from a portionof the internal chamber above the rib. In particular, the plungerdiameter d of the sealing rib 1272 may be interference fit to the pelletdiameter (e.g., slightly greater than the diameter of the body 1205 atthe pellet region 1204), such that the plunger 1210, via rib 1272,engages the pellet region 1204 during actuation of the plunger. In thismanner, the plunger 1210 may fluidically isolate the pellet region 1204from the tapered region 1203 and the collection region 1202 whenactuating the plunger. The sealing rib 1272 may be molded with and madefrom the same, generally-rigid material as the plunger.

In some embodiments, the plunger 1210 may further include a plunger seal1290 attached to the plunger above the sealing rib 1272 (e.g., thesealing rib 1272 may be disposed between the plunger seal 1290 and thepoint 1217. The plunger seal 1290 may define a diameter that is greaterthan or equal to the diameter of the sealing rib 1272 and the diameterof the pellet region 1204. As described herein, the plunger seal 1290may seal the internal chamber 1211 of the body at the pellet region 1204to prevent leakage of the density cushion when the sealing rib 1272emerges from the first distal end 1206 of the body 1205 when the plunger1210 is fully depressed.

In some embodiments, the plunger 1210 may define a recess 1273 in thelongitudinal member 1216 in which a portion of the plunger seal 1290 isdisposed. The recess 1273 may include a first shoulder 1274 and a secondshoulder 1275 which may abut either side of the plunger seal 1290 toretain the plunger seal within the recess and may extend radially fromthe inner diameter of the recess to a diameter of the longitudinalmember 1216. In some embodiments, further ridges (also referenced hereinas chamfers) (shown in FIG. 260) or changes in diameter may be used toretain the plunger seal 1290.

In some embodiments, the plunger 1210 may further define a throughpassage 1276 in the recess 1273 through which a corresponding lockingarm 1277 of the plunger seal 1290 may extend to prevent rotational oraxial movement of the plunger seal. In some embodiments, the plungerseal 1290 may be overmolded onto the plunger 1210 such that the lockingarm 1277 is fixedly and permanently formed within the through passage1276. In some embodiments, the plunger seal 1290 may be formed from asofter material (e.g., an elastomeric material) than the plunger 1210 toprovide a further seal against the wall 1209 when the plunger isdepressed. The plunger seal 1290 may be made from an elastomer suchsilicone or thermoplastic elastomers just as with coupling member 1240.In some embodiments, the material of the plunger seal 1290 should becompliant (e.g., TPE with a 50 Shore A durometer). The plunger seal 1290may mitigate molding defects in the more rigid plunger 1210 (e.g.,imperfections in the seal of the sealing rib 1272. In some embodiments,the plunger seal 1290 may deform more easily than the rib 1272 andrequire less force to move along the wall 1209. The plunger seal 1290may include chamfered edges near the shoulders 1274, 1275 for easieractuation.

In some embodiments, the longitudinal member 1216 of the plunger mayinclude a further tapered region 1278 above the recess 1273 (e.g., therecess 1273 is disposed between the point 1217 and the further taperedregion 1278) such that the portion of the longitudinal member 1216 abovethe further tapered region 1278 is wider. The further tapered region1278 and the longitudinal member 1216 on the larger side of the furthertaper may define a diameter that is substantially greater than thepellet region 1204.

In some embodiments, the longitudinal member 1216 may include a step1279 which may help to reduce the required actuation force of theplunger 1210 because of the relatively narrower diameter of the stepcompared to the surrounding body of the plunger.

With reference to FIGS. 259, 262-265, and 267, a second distal end 1219of the plunger, opposite the point 1217, may include a gripping portion1280 for allowing the user to depress (e.g., move the plunger axially inthe longitudinal direction) and/or twist (e.g., rotate the plunger aboutthe longitudinal axis, for example, to release the support member 1215and retainer 1232) the plunger 1210. The gripping portion 1280 may begenerally cylindrical (e.g., as shown in FIG. 1210, such that the usercan rotate the plunger 1210 by rolling the gripping portion 1280 betweentheir fingers. In some embodiments, the gripping portion 1280 may beasymmetrical (e.g., oblong like the tab 1022 detailed above).

With reference to FIGS. 224-239 and 269-272, the gripping portion 1280of the plunger 1210, which may be sealed within the separation container1200, may be manipulated through the flexible sealing member 1225 toprevent leakage or contamination. The flexible sealing member 1225 maybe made of an elastomeric material, and in some embodiments, theflexible sealing member 1225 may be made of an at least partiallytransparent material. In some embodiments, the flexible sealing member1225 may be retained by a cap 1230 that is structured and functionssubstantially the same as the caps (e.g., caps 130, 1030) describedherein, with an opening 1231 (shown in FIG. 224) through which theflexible sealing member 1225 may extend. The cap 1230 may then threadonto the body 1205 to secure a flange 1281 of the flexible sealingmember therebetween. In some embodiments depicted herein (e.g., as shownin FIG. 225, 228, 231-233, 236-237), the flexible sealing member 1225 isdepicted as being transparent to allow visibility of the plunger 1210therein. However, in some embodiments, the flexible sealing member 1225may be transparent, opaque, or partly transparent or opaque.

With reference to FIGS. 269-271, the flexible sealing member 1225 mayinclude a top 1282, which the user may depress to also depress theplunger 1210 (shown in FIG. 224). The top 1282 and flange 1281 may beconnected by a wall 1283 of the flexible sealing member 1225. Turning toFIGS. 273-276, upon actuation by the user, the top 1282 may translatedownwardly towards the flange 1281 and remainder of the separationcontainer to cause the axial movement of the plunger 1210. In someembodiments, the top 1282 may collapse slightly around the user'sfinger. As the top 1282 and flange 1281 move closer to each other thewall 1283 collapses and hinges to allow the top 1282 to remain incontact with the plunger. In some embodiments, the wall 1283 deforms atleast partially outward to allow the top 1282 to depress the plunger1210 to a position completely flush with the cap 1230.

Referring to FIGS. 269-274, in some embodiments, wall 1283 comprises aplurality of segments 1284, 1285, 1286, 1287, which may be vertical(e.g., the vertical segments 1285, 1287) or non-vertical (e.g., thenon-vertical segments 1284, 1286). In some embodiments, the wall 1283may define an at least partially inwardly concave shape (e.g., thesemi-rounded shape shown in FIG. 271) to cause the wall to deflect outof the path of the plunger. For example, in some embodiments, the wallmay include a vertical segment 1285 adjacent two non-vertical segments1284, 1286. The vertical wall segment 1285 may be generally concentricabout the longitudinal axis of the plunger 1210 with the surface of thevertical wall segment 1285 defining an at least partial cylinder sharinga common axis with the plunger. The two non-vertical segments 1284, 1286may each be angled radially inwardly from their respectivecircumferential junctions with the vertical segments 1285. In suchembodiments, the travel distance of the plunger 1210 and the deformationrange of the flexible sealing member 1225 may be greater than anequivalently tall bellows design.

In the embodiment shown in FIG. 271, the top 1282 is connected to afirst non-vertical wall segment 1284 that is angled downwardly andoutwardly from the top. The first non-vertical wall segment 1284 is thenconnected to a first vertical wall segment 1285. The first vertical wallsegment 1285 is then connected to a second non-vertical wall segment1286 that is angled downwardly and inwardly from the first vertical wallsegment. The second non-vertical wall segment 1286 is then connected toa second vertical wall segment 1287, which abuts the flange 1281. Insome embodiments, the wall segments may smoothly transition between eachother. In some embodiments, the flexible sealing member (e.g., flexiblesealing members 125, 1025) of any other embodiment may be used in placeof the flexible sealing member 1225.

In some embodiments, the wall 1283 may hinge about the non-vertical wallsegments 1284, 1286 with the vertical wall segments 1285, 1287 remainingsubstantially vertical, such that the first vertical wall segment 1285moves outwardly, while the non-vertical wall segments 1284, 1286 hingeand the top 1282 moves downwardly during operation. For example, FIGS.273-276 illustrate an embodiment of the flexible sealing member 1225 inan actuated position. In the depicted embodiment, the top 1282 of theflexible sealing member 1225 has been pressed downwardly with the wall1283 hinging about the junction between the first non-vertical wallsegment 1284 and the first vertical wall segment 1285, with portions ofthe top 1282 and/or wall 1283 optionally elongating with the force ofthe actuation. In some embodiments, as discussed herein, the flexiblesealing member 1225 may deform sufficiently far to press the top of theplunger 1210 to a position parallel to, above, below, parallel or below,or parallel or above, a plane of the cap 1230, a plane of the top of thebody 1205, and/or a plane of the flange 1281.

Referring back to FIGS. 224-239, the separation container 1200 mayinclude a rheological control member 1242, which may be structured andoperate according to any of the embodiments discussed herein. Forexample, in some embodiments, the rheological control member 1242, body1205, and plunger 1210 may be structured and cooperate in substantiallythe same manner as the rheological control member 1042, body 1005, andplunger 1010 of FIG. 165 or FIG. 213, and may include the gasket 1043and corresponding structure and operation described therewith. In someother embodiments, the rheological control member 1242 may be structuredand operate as shown and described with respect to the embodiments ofFIGS. 14-31, FIGS. 32-49, or FIGS. 50-67.

Unless otherwise stated, the separation container 1200 may operate inthe same manner, may have the same properties, and may be made with thematerials and configurations of any embodiment described herein. Theseparation container 1200 of the sixth embodiment depicts a non-buoyantplunger 1210 that is retained by a retainer 1232 as described above. Thedepicted separation container 1200 also includes a rheological controlmember 1242 as described herein. In addition, the depicted separationcontainer 1200 may engage an end cap 1250 for securing the seal 1220(e.g., a foil or other sealing membrane) to the body 1205 and supportingthe separation container during centrifugation. The separation container1200 may further include a coupling member 1240 for coupling the body1205 with a sample collecting vessel 135, 1038, which may be used in asimilar manner to the sample collecting vessels 135, 1038 describedherein.

During assembly, the separation container 1200 may be assembled in thefollowing ordered steps: (1) add the density cushion to the internalchamber 1211; (2) connect the plunger 1210 with a retainer 1232; (3)insert the rheological control member 1242 onto the plunger 1210; (4)slide the gasket 1043 onto the plunger 1210 beneath the rheologicalcontrol member 1242 (for embodiments using a gasket); (5) engage theretainer 1232 with the slots 1265 in the body 1205, while also formingan interference or slip fit between the rheological control member 1242and the body 1205; (6) insert the flexible sealing member 1225 into theopening 1231 in the cap 1230; and (7) secure the flexible sealing member1225 and cap 1230 onto the body 1205 while encapsulating the plungerwithin the separation container (e.g., seal the separation containereither before or after inserting a sample for testing). An interferencefit may be created by inserting rods into the gaps between the retainer1232 and the plunger 1210 to press the rheological control member 1242downwardly. In some embodiments the seal 1220 may be attached to thebody 1205 prior to adding the density cushion. In accordance with someembodiments discussed herein, the rheological control member may beinitially inserted with an interference fit before being released duringcentrifugation by the outward deformation of the wall 1209 of the body1205.

During testing, the separation container 1200 may be operated in thefollowing ordered steps: (1) To add the lysed sample to the tube, thecap 1230 and flexible sealing member 1225 may be removed; (2) the samplemay then be transferred to the body 1205, with the rheological controlmember 1242 preventing bulk mixing of the sample with the densitycushion by being positioned between the two fluids; (3) the separationcontainer may be optionally pre-spun before adding the sample to moveany density cushion that may have migrated to the top chamber duringshipping or storage. The rheological control member 1242 may beconstructed for a small clearance between it and the wall 1209 of thebody 1205 and may include annular shoulders (e.g., shoulders 1061, 1062)when not being centrifuged as discussed above (e.g., to limit thedownward movement of the rheological control member). In someembodiments, the rheological control member 1242 may also be constructedso that it does not interact with or reach the tapered region 1203 ofthe body 1205.

The testing may further include: (4) during addition of the sample therheological control member 1242 may float if optionally not interferencefit, while still providing mixing protection. The rheological controlmember 1242 may also have a recessed ring in the top portion, which cantrap any settling components such as resin or other particulates. Theseparticles may stay in the recessed area as the layering aid floats upthe tube either during sample addition or centrifugation. The testingmay further include: (5) after the sample is added, the cap 1230 andflexible sealing member 1225 may be placed back on the tube; and (6) theentire separation container 1200 may be placed in the centrifuge andspun. In embodiments where the rheological control member 1242 is sealedagainst the body 1205 and plunger 1210, the sample may be added directlyto an internal chamber 1211 with a lytic agent and the entire separationcontainer 1200 can be vortexed to lyse the sample before thecentrifugation step.

In operation, the process for expressing the pellet is the sameactuation and foil piercing process described herein with respect toeach other plunger embodiment, and in embodiments using a retainer 1232,an additional step of applying horizontal, rotational pressure to thegripping portion 1280 of the plunger 1210 may be used to disconnect theplunger from the retainer. With reference to FIGS. 225-230, theseparation container 1200 is shown in a storage or loaded position(depending on whether sample has been introduced to the body 1205) inwhich the plunger 1210 is attached to and retained by the retainer 1232.FIGS. 231-235 show the separation container 1200 whose plunger 1210 hasbeen released from the retainer 1232, but the plunger has not yetpierced the seal 1220. In the position shown in FIGS. 231-235, thesealing rib 1272 has abutted the wall 1209 of the body 1205 at thejunction between the tapered region 1203 and the pellet region 1204 whenthe point 1217 contacts the foil. Turning to FIGS. 236-239 and 275-276,the plunger 1210 is shown fully depressed with the rib 1272 havingpassed through the first distal end 1206 of the body 1205, the pellethaving been fully expressed, and the plunger seal 1290 preventingleakage of the fluid (e.g., density cushion) thereabove. For example, insome embodiments, the distance between the sealing surfaces of the rib1272 and the plunger seal 1290 may be less than the length of the pelletregion 1204 to prevent leakage of the density cushion or contaminationof the sample. Unless otherwise stated, features having the samereference numeral, name, structure, or purpose in the assembly may beinterchanged with one another in any of the embodiments described hereinand the present inventors specifically contemplate each possiblepermutation of structures and features.

Seventh Embodiment

With reference to FIGS. 277-344, a seventh embodiment of the separationcontainer 1300 is shown. With reference to FIG. 277, an exploded view ofthe separation container 1300 is shown depicting a body 1305 having aseal 1320 to close an opening 1312 (shown in FIG. 280) in one end and acap 1330 and flexible sealing member 1325 at the other. The separationcontainer 1300 may include a plunger 1310 having a plunger seal 1390, arheological control member 1342, and a retainer 1332 attached theretofor ensuring smooth operation and reducing contamination of the samplepellet as described below. The separation container 1300 may furtherengage an end cap 1350 for supporting the body 1305 and seal 1320 duringcentrifugation, and the separation container may include a couplingmember 1340 for coupling and sealing the body to a sample collectingvessel (e.g., the sample collecting vessels 135, 1038 described herein).

With reference to FIGS. 278-282, assembled views of the separationcontainer 1300 are shown with an end cap 1350. In some embodiments, theseparation container 1300 may engage the end cap 1350 to support theseal 1320 and body 1305 during centrifugation to allow the separationcontainer to be adaptable to many different centrifuge cups while alsoimproving manufacturability and quality. The end cap 1350 may hold theseal 1320 in place during induction welding in instances in which theseal is induction welded to the tube. If the seal 1320 is heat sealed,the seal may be attached before the end cap 1350 is applied. In each ofthe embodiments discussed herein, the end cap 1350 may support the seal1320 from distending and rupturing during centrifugation. In someembodiments, the seal 1320 may be connected to the body 1305 withsufficient strength to prevent the seal from opening under the force ofcentrifugation without an end cap 1350.

In some embodiments, the end cap 1350 may be a centrifuge adaptorconfigured to stably support the separation container 1300 in acentrifuge. The end cap 1350 may include a tapered portion 1351, whichmay be solid or include one or more ribs 1352 with gaps therebetween. Insome embodiments, a portion of the ribs 1352 may define the taperedportion 1351. The end cap 1350 may include a substantially flat distalend 1353 and may include a flange 1354 and/or a concentric wall 1358 atan opposite end to assist with alignment of the end cap 1350 in therespective axial and radial directions. The flat distal end 1353 may beconfigured to engage the flat or rounded bottoms of various types andmodels of centrifuge cup, and the distal end 1353 may be narrower thanthe flange 1354 and/or concentric wall 1358 at the opposite end with thetapered portion 1351 narrowing the diameter of the end cap between thetwo ends. In some embodiments, the tapered portion 1351 may beconfigured to engage the walls of conical-shaped centrifuge cups. Thus,the end cap 1350 may be structured to engage different models andstructures of centrifuge without needing different adaptors orsacrificing stability.

In some embodiments, the body 1305 may include a flange 1355 that isconfigured to engage the flange 1354 and/or concentric wall 358 of theend cap 1350. In some embodiments, the flange 1355 may be disposedcircumferentially about the tapered region 1303 of the body 1305 andoriented towards the flange 1354 of the end cap 1350, as shown in theembodiment of FIGS. 277-280. In some embodiments, the flanges 1354, 1355may be removably or semi-permanently attached to each other. Forexample, in some embodiments, a frictionally-retained interference fit,a threaded connection (not shown), snap connection (not shown), or otherremovable engagement may be formed between the body flange 1355 and theend cap flange 1354. In some embodiments, the flanges 1354, 1355 may beadhered, welded, molded, or otherwise semi-permanently fused to eachother, such that the bond between the flanges must be mechanicallybroken to remove the end cap 1350. In some embodiments, the end cap 1350may be press fit over the coupling member 1340, such that the end cap isfrictionally retained and does not need to be welded or threaded to thebody 1305. In each instance, the end cap 1350 may be optionally weldedto the body 1305.

In some embodiments, the end cap 1350 may include a hex-shaped recess1357 in the distal end 1353. The recess 1357 may be used to hold theseparation container 1300 (e.g., by standing the separation containerand end cap 1350 on a vertical post (not shown)) when not in use, whenfilling the container, or during centrifugation. In some embodiments, apost or other rigid object (not shown) may be inserted into the recess1357 to assist with removal of the end cap 1350 after centrifugation.For example, in embodiments in which the end cap 1350 has its flange1354 semi-permanently attached to the flange 1355 of the body, the postor other rigid object may be a corresponding hex shape, such that thepost or other rigid object may be inserted into the recess 1357 and maybe used to twist or torque the semi-permanent connection apart. Themechanical separation of the end cap 1350 with a tool (e.g., the post orother rigid object referenced above) may reduce agitation of the sampleand prevent inadvertent contamination.

In some embodiments, the end cap 1350 may further include an internalplatform 1356 (also referred to herein as a “support surface”) againstwhich the first distal end 1306 of the pellet region 1304 may rest,either with the body 1305 touching the platform 1356 or with the seal1320 sandwiched therebetween. The platform 1356 may support the seal andprevent inadvertent rupture before the user is ready to express thefinal concentrated pellet, including during manufacturing, assembly,packaging, loading, and centrifugation. The internal platform 1356 maydefine a substantially flat, planar surface in the internal cavity ofthe end cap 1350 against which the seal 1320 during centrifugation. Inaddition, the platform 1356 of the end cap 1350 allows a full sized seal1320 to be attached to the entire surface area of the distal end of thepellet region 1304 providing for improved manufacturability and animproved seal between the seal 1320 and body 1305.

With reference to FIGS. 307-316, an embodiment of the end cap 1350 isshown. In the depicted embodiment, the end cap 1350 further comprisesprojecting ribs 1359 extending into the interior cavity of the end cap.The projecting ribs 1359 may include one or more ribs, and in someembodiments, a plurality of projecting ribs 1359 are spacedcircumferentially about the end cap relative to an axis A (shown in FIG.311) of the end cap. With reference to FIGS. 280-281, in someembodiments, the projecting ribs 1359 are configured to deform thecoupling member 1340 to allow air trapped at the end of the internalcavity of the end cap 1350 adjacent the platform 1356 to escape when thedistal end 1306 of the body 1305 is inserted into the end cap 1350. Insuch embodiments, the ribs 1359 deform the coupling member 1340 to allowair to escape between ribs and/or between the ribs and the deformedsurface of the coupling member (e.g., the first distal sealing surface1343 of the coupling member). In some embodiments, the projecting ribs1359 are disposed at a shelf joining the first wall 1346 and the secondwall 1347 of the end cap 1350. Once the first distal end 1306 of thebody is inserted completely into the end cap 1350, the coupling member1340 may seal against the end cap 1350 and prohibit furthercommunication between the seal 1320 and the external environment.

With reference to FIGS. 277, 279, 283-288, 317-319, and 330-334, acoupling member 1340 may be disposed on the body 1305 at or near thepellet region 1304 to facilitate coupling of the separation container1300 to a sample collecting vessel (e.g., the sample collecting vessels135, 1038 detailed herein) and/or to provide a further seal within theend cap 1350 to prevent leakage of the contents of the body 1305. Thecoupling member 1340 may include one or more circumferential ridges 1341to provide a seal against the sample collecting vessel. The couplingmember 1340 may further include a first, distal sealing surface 1343 anda second, upper sealing surface 1344, which may be pointed (e.g., likeridges 1341) or flat. The sealing surfaces 1343, 1344 and the ridges1341 may progressively decrease in their diameter when travellingtowards the first distal end 1306 of the pellet region 1304. Forexample, the first sealing surface 1343 may be the narrowest sealingsurface of the coupling member 1340 and the second sealing surface 1344may be the widest sealing surface of the coupling member with each ridge1341 therebetween progressively increasing in diameter, which mayfacilitate insertion of the separation container into the samplecollecting vessel while providing a strong seal.

With reference to FIG. 280, in some embodiments, the second sealingsurface 1344 may be wider than the inner diameter of a sample collectingvessel (not shown) (e.g., the sample collecting vessels 135, 1038 shownherein) and the second sealing surface 1344 may include a chamfer 1345for engaging the upper edge of the sample collecting vessel. The chamfer1345 may either create a seal against the upper rim of the samplecollecting vessel or may compress the second sealing surface 1344 inwardto fit the second sealing surface within the sample collecting vessel,while reducing the force required for engagement. In some embodiments,any other connector or coupling may be used to engage the body 1305 witha sample collecting vessel. The other connector or coupling may beattached to either the body 1305 or the sample collecting vessel or maybe a separate device.

The end cap 1350 may include a narrow, first wall 1346 near or adjacentto the platform 1356 and may include a wider, second wall 1347 above thefirst wall opposite the platform. The first wall 1346 and the secondwall 1347 (the inner and/or outer surfaces) may be parallel to the wallof the pellet region 1304 or may be tapered inwardly slightly such thatthe distal end closest to the platform 1356 is narrower than theopposite end for either wall segment 1346, 1347. The first wall 1346 andsecond wall 1347 may define diameters that are less than or equal to thediameters of the respective first sealing surface 1343 and secondsealing surface 1344 at least at the same respective axial positionsabove the platform 1356. In the embodiment of FIGS. 280-281, the firstsealing surface 1343 is shown overlapping with the first wall 1346 ofthe end cap 1350 because the depicted coupling member 1340 has a widerdiameter than the depicted end cap 1350 at this axial location. Inreality, the first sealing surface 1343 deforms to fit within the distalend of the end cap 1350 in such an embodiment, and no overlap will occurin the depicted embodiment.

With continued reference to FIG. 280, the body 1305 may include one ormore circumferential ribs 1348, 1349 that provide structural rigidity tothe connection between the body and the coupling member 1340. Forexample, in the embodiment shown in FIG. 280, the body includes twocircumferential ribs 1348, 1349, one disposed proximate the taperedregion 1303 of the body and one disposed proximate the first distal end1306 of the pellet region 1304. In the embodiment shown in FIGS. 280 and332, a first circumferential rib 1348 is embedded in a correspondingrecess 1360 in the first sealing surface 1343 of the coupling member1340. Similarly, in the embodiment shown in FIGS. 280 and 332, a secondcircumferential rib 1349 is embedded in a corresponding recess 1361 inthe second sealing surface 1344 of the coupling member 1340.

The coupling member 1340 and the body 1305 may be attached viaovermolding, elastically stretching the coupling member 1340 over thebody 1305, or fusing multiple pieces of the coupling member 1340together around the body 1305. In some embodiments, the coupling member1340 may be made of a compliant material such as elastomer. The couplingmember 1340 may be overmolded onto the body 1305. In some embodiments,the coupling member 1340 may be made from silicone or thermoplasticelastomers such as Medalist® MD-12140. In some embodiments, theseparation container opening 1312 may be sealed by the seal 1320, by theend cap 1350 pressing on the seal, and by the engagement of the end cap1350 and the coupling member 1340.

With reference to FIGS. 319-334, the body 1305 may also include one ormore vertical ribs 1362, and the coupling member 1340 may include one ormore corresponding recesses 1363 for receiving and engaging the verticalribs 1262. In some embodiments, the body 1305 may include a base flange1364 at the distal end of the pellet region 1304 for retaining thecoupling member 1340 with its upper surface and engaging the seal 1320with its lower annular surface (e.g., via sonic welding), with theannular surface surrounding the opening 1312 (shown in FIG. 280).

An end of the coupling member 1340 opposite the base flange 1364 mayfurther abut a portion of the outer surface of the tapered region 1303,such that the combined upward retention from the base flange and thedownward retention from the tapered region may retain the couplingmember 1340 in a stable position (e.g., the position shown in FIG. 318).In some embodiments, the coupling member 1340 may be disposed betweenthe flange 1355 on the body 1305 in the tapered region 1303 and the baseflange 1364 such that the coupling member 1340 fits within the end cap1350 and sample collection vessels described herein to prevent fluid(including the sample) from leaking out of the separation container. Thecoupling member 1340 may include bi-directional (e.g., perpendicular)recesses (e.g., recesses 1360, 1361, 1363) to engage the correspondingbi-directional ribs (e.g., ribs 1348, 1349, 1362) of the body andprevent both axial and rotational movement of the coupling member.

As discussed in connection with other embodiments herein, the body 1305may include a first distal end 1306, a second distal end 1307, a taperedregion 1303, a pellet region 1304, a collection region 1302, a widenedregion 1308, and a wall 1309, each of which may operate and may bestructured as described in each embodiment herein. The separationcontainer 1300 may further include a retainer 1332 attached to the body1305 in the widened region 1308. In some embodiments, with reference toFIGS. 319, 320, 322, 323, 327, the widened region 1308 of the body 1305may include one or more supporting projections 1365 which may receiveand secure the retainer 1332 on a distal end thereof. In someembodiments, the inner diameter of the body 1305 with the projection1365 in the widened region 1308 is the same as the diameter of thecollection region 1302. The retainer 1332 may be otherwise structuredand operable as described above with respect to the retainers 1032, 1232to secure the plunger 1310 in a predetermined vertical position (e.g., aposition in which the upper edge of the retainer 1332 is coplanar withthe second distal end 1307 of the body 1305). For example, withreference to FIG. 259, the plunger 1310 may include a support member1315 having the features and structure shown and described in connectionwith the support member 1015, 1215 of the plunger 1010, 1210 above, andthe retainer 1332 may include retaining members 1333 and correspondingfeatures having the features and structure shown and described inconnection with the retainer 1032, 1232 of the plunger 1010, 1210.

With reference to FIGS. 296-306, the plunger 1310 is shown in accordancewith some embodiments discussed herein. The plunger 1310 may include alongitudinal member 1316 coming to a conical point 1317 at a firstdistal end 1318. The plunger may include a piercing section 1370defining a cylindrical portion of the longitudinal member 1316 whichterminates with a taper at the point 1317. The piercing section 1370 mayoperate in substantially the same manner as the blade 160 or point 117discussed herein, and the piercing section 1370 and point 1317 may benarrower than the longitudinal member 1316 or the remaining portions ofthe plunger 1310 to allow for collection of additional sample in thepellet region 1304. In this manner, the relative diameters and sizes ofthe components shown in the figures are intended to be accurate asexample embodiments (e.g., the piercing section 1370 is depictednarrower than the remainder of the longitudinal member 1316).

In some embodiments, the plunger 1310 may include at least one sealingrib 1372 formed circumferentially about the longitudinal member 1316,and may include a tapered region 1371 between the sealing rib 1372 andthe piercing section 1370. This sealing rib 1372 may be a generallycircular sealing surface that seals uniformly about the plunger. Thesealing rib 1372 may be configured to engage the wall 1309 of the body1305 and seal a portion of the internal chamber 1311 below the rib 1372from a portion of the internal chamber above the rib. In particular, thediameter d of the sealing rib 1372 may be interference fit to the pelletdiameter (e.g., slightly greater than the diameter of the body 1305 atthe pellet region 1304), such that the plunger 1310, via rib 1372,engages the pellet region 1304 during actuation of the plunger. In thismanner, the plunger 130 may at least partially fluidically isolate thepellet region 1304 from the tapered region 1303 and the collectionregion 1302 when actuating the plunger. In some embodiments, the sealingrib 1372 may define a diameter that is equal to or greater than adiameter of the pellet region 1304. In some embodiments, the sealing rib1372 may define a diameter that is equal to or less than a diameter ofthe pellet region 1304. The sealing rib 1372 may be molded with and madefrom the same, generally-rigid material as the plunger.

In some embodiments, the plunger 1310 may include a plunger seal 1390attached to the plunger in addition to or instead of the sealing rib1372. In some embodiments, the sealing rib 1372 may be disposed betweenthe plunger seal 1390 and the point 1317 at the first distal end 1318.The plunger seal 1390 may define a plunger diameter that is greater thanor equal to the diameter of the sealing rib 1372 and greater than orequal to the diameter of the pellet region 1304. As described herein,the plunger seal 1390 may seal the internal chamber 1311 of the body atthe pellet region 1304 to prevent leakage of the density cushion whenthe portions of the plunger between the plunger seal and the point 1317emerge from the opening 1312 (shown in FIG. 280) at the first distal end1306 of the body 1305 when the plunger 1310 is fully depressed. In someembodiments, a volume of sample is configured to be retained between theplunger seal 1390, the wall of the separation container body 1305 (e.g.,in the pellet region 1304 during actuation), the first distal end 1306of the body 1305, and a portion of the longitudinal member 1316 betweenthe point 1317 and the plunger seal 1390. The volume is retained uponactuation of the plunger 1310 when the plunger seal 1390 engages thewall 1309 at the pellet region 1304. As described herein, variousdiameters of the plunger 1310 may be taken radial to an axis (e.g., axisA shown in FIG. 279) extending between the first distal end 1318 and thesecond distal end 1319 of the plunger. In such embodiments, engagementof the plunger seal 1390 with the wall 1309 (e.g., at the pellet region1304) may divide the internal chamber 1311 into at least twosub-chambers above and below the plunger seal.

In some embodiments, the plunger 1310 may define a recess 1373 in thelongitudinal member 1316 in which a portion of the plunger seal 1390 isdisposed. The recess 1373 may include a first shoulder 1374 and a secondshoulder 1375 which may abut either side of the plunger seal 1390 toretain the plunger seal within the recess and may extend radially fromthe inner diameter of the recess to a diameter of the longitudinalmember 1316. In some embodiments, further ridges (also referenced hereinas chamfers) (shown in FIG. 302) or changes in diameter may be used toretain the plunger seal 1390.

In some embodiments, the plunger 1310 may further define a throughpassage 1376 in the recess 1373 of the longitudinal member 1316 throughwhich a corresponding locking arm 1377 of the plunger seal 1390 mayextend to prevent rotational or axial movement of the plunger seal. Insome embodiments, the plunger seal 1390 may be overmolded onto theplunger 1310 such that the locking arm 1377 is fixedly and permanentlyformed within the through passage 1376. In some embodiments, the plungerseal 1390 may be formed from a softer material (e.g., an elastomericmaterial) than the plunger 1310 to provide a further seal against thewall 1309 when the plunger is depressed. In some embodiments, theplunger seal may be integral with the plunger 1310. The plunger seal1390 may be made from an elastomer such silicone or thermoplasticelastomers as described herein with respect to the coupling member 1340.In some embodiments, the material of the plunger seal 1390 should becompliant (e.g., TPE with a 50 Shore A durometer). The plunger seal 1390may mitigate molding defects in the more rigid plunger 1310 (e.g.,imperfections in the seal of the sealing rib 1372. In some embodiments,the plunger seal 1390 may deform more easily than the rib 1372 andrequire less force to move along the wall 1309. The plunger seal 1390may include chamfered edges near the shoulders 1374, 1375 for easieractuation.

In some embodiments, the longitudinal member 1316 of the plunger mayinclude a further tapered region 1378 (also referred to as a “shoulder”herein) above the recess 1373 (e.g., the recess 1373 is disposed betweenthe point 1317 and the further tapered region 1378) such that theportion of the longitudinal member 1316 above the further tapered region1378 is wider. The further tapered region 1378 and the longitudinalmember 1316 on the larger side of the further taper may define adiameter that is substantially greater than the pellet region 1304. Insome embodiments, the distance between the further tapered region 1378and the point 1317 of the plunger 1310 may be less than or equal to anaxial length of the pellet region 1304, such that the further taperedregion 1378 is configured to impinge on the wall 1309 proximate thejunction between the tapered region 1303 of the body 1305 and the pelletregion 1304 of the body before the plunger seal 1390 completely exitsthe first distal end 1306 of the body and before fluid above the plungerseal can escape. In some embodiments, the impingement of the furthertapered region 1378 (also referred to as a shoulder) may define themaximum axial displacement of the plunger 1310. In some embodiments, anaxial distance between the plunger seal 1390 and the tapered region 1378and an axial length of the pellet region 1304 are configured such thatthe plunger seal 1390 remains at least partially within the body 1305 atthe maximum displacement of the plunger 1310. In some embodiments, anaxial distance between the plunger seal 1390 and the first distal end1318 of the plunger is less than or equal to an axial length of thepellet region 1304 such the plunger seal 1390 is configured to engagethe pellet region 1304 of the body 1305 before the plunger 1310 opensthe seal 1320 during actuation. In some embodiments, the further taperedregion 1378 may define an angle that matches the angle of the taperedregion 1303 according to any of the embodiments described herein.

In some embodiments, the longitudinal member 1316 may include a step1379 which may help to reduce the required actuation force of theplunger 1310 because of the relatively narrower diameter of the stepcompared to the surrounding body of the plunger.

With reference to FIGS. 296, 299-301, 304-305, a second distal end 1319of the plunger, opposite the point 1317 at the first distal end 1318,may include a gripping portion 1380 for allowing the user to depress(e.g., move the plunger axially in the longitudinal axis extendingbetween the first distal end 1318 and the second distal end 1319) and/ortwist (e.g., rotate the plunger about the longitudinal axis, forexample, to release the support member 1315 and retainer 1332) theplunger 1310. In some embodiments, the gripping portion 1380 may bespaced from the second distal end 1319 (e.g., as shown in FIG. 301). Insome embodiments, the gripping portion 1380 may be substantiallyrectangular. For example, with reference to FIG. 305, the grippingportion 1380 may define a rectangular or square cross section in a planeperpendicular to the axis of the longitudinal member 1316. In someembodiments, the gripping portion may be generally cylindrical. In someembodiments, the gripping portion may be asymmetrical. In someembodiments, the gripping portion may include knobs or projections tofacilitate rotation of the plunger about the axis of the longitudinalmember.

In some embodiments, the retainer (also referred to as a “collar”herein) 1332 may be configured to engage the plunger 1310 to preventaxial displacement of the plunger. In some embodiments, the retainer1332 may comprise an annular wall 1337 having engagement features (e.g.,retaining members 1333) defined thereon. In some embodiments, theretainer may comprise features (e.g., walls, grooves, or projections)integral with the body 1305 of the separation container 1300 configuredto engage one or more portions of the plunger 1310, such that theretainer and the body define a single piece. In some embodiments, theretainer 1332 may be separately engaged with the body 1305 (e.g., viapress fit).

With reference to FIGS. 296, 299, 300, 301, 303, and 339-344, theretainer 1332 and plunger 1310 may have a retention mechanism thatallows the plunger to be supported by the retainer during centrifugationbut also actuatable by a user after separation. In particular, in someembodiments, the retainer 1332 may support the plunger 1310 under axialloads (e.g., loads along the axis A shown in FIG. 279) of at leastthree-thousand times the force of gravity, while the plunger 1310 mayengage and disengage with the retainer 1332 by rotating the plunger 1310about the axis A shown in FIG. 279. In particular, the retainer 1332includes one or more retaining members 1333 that engage correspondingsupport member 1315 on the plunger 1310. The support member 1315 mayinclude one or more locking members 1314 that engage the respectiveretaining members 1333 of the retainer 1332. In the depicted embodiment,the retainer 1332 includes two retaining members 1333 and the plunger1310 includes two locking members 1314 each separated from the other by180 degrees about the longitudinal axis of the plunger 1310.

In the depicted embodiment, the retaining members 1333 may include asupport projection 1334 extending from the annular wall 1337 of theretainer 1332 with a stop wall 1336 at one end of the supportprojection. The support projection 1334 may further include one or morelocking tabs 1335. With reference to FIG. 303, the locking members 1314may include a substantially C-shaped receiving area defined by a lowerwall 1396, a lateral wall 1397, an upper wall 1398, and a lip 1399.These features may combine to receive and engage the correspondingretaining member 1333 of the retainer 1332. For example, the supportprojection 1334 (shown in FIGS. 339-344) of the retaining member 1333may be received in the locking member 1314 (shown in FIG. 303) with anupper surface of the lower wall engaging a lower surface of the supportprojection 1334. The locking tab 1335 (shown in FIGS. 339, 342, and 344)and/or the lip 1399 may deflect when the retaining member 1333 isengaged with the locking member 1314 such that the locking tab 1335 maybe disposed in a space between the lateral wall 1397, the upper wall1398, and the lip 1399 to retain the plunger 1310.

In some embodiments, the inner, annular wall 1337 of the retainer 1332may define a circumferential direction in a plane spanning the centralopening and oriented about the central opening and an axial directionoriented perpendicular to the plane (e.g., along the axis A shown inFIG. 279). In some embodiments, the at least one support projection 1334may define a circumferential wall that is longer in the circumferentialdirection than in the axial direction (e.g., as shown in FIGS. 339-344).In some embodiments, the stop wall 1336 may be configured to preventrotation of the plunger in a clockwise direction or a counterclockwisedirection in an instance in which the at least one support projection1334 is engaged with the at least one locking member 1314 of the plunger1310. In some embodiments, the stop wall 1336 may extend from a firstsurface of the at least one support projection 1334 and may preventrotation of the plunger in a clockwise direction or a counterclockwisedirection about the axis A. In some embodiments, the retainer 1332 mayinclude two retaining members 1333 disposed diametrically opposite oneanother relative to the annular wall 1337. In some embodiments, thesupport member 1315 may include two locking members 1314 disposeddiametrically opposite one another relative to the longitudinal axis ofthe plunger 1310.

In some embodiments, the at least one retaining member 1333 may includeat least one locking tab 1335 extending from a first surface of the atleast one support projection 1334. The at least one locking tab 1335 maybe configured to engage the at least one locking member 1314 of theplunger 1310 to releasably retain the plunger by increasing a forcerequired to rotate the plunger about the longitudinal axis A when the atleast one locking tab 1335 is engaged with the at least one lockingmember 1314 of the plunger 1310. In some embodiments, the at least onelocking member 1314 defines a C-shaped wall configured to be disposed onboth sides of the circumferential wall relative to the axial directionin an instance in which the at least one locking member and the at leastone retaining member are engaged, and the C-shaped wall may comprise thelower wall 1396, lateral wall 1397, upper wall 1398, and/or the lip 1399discussed herein. In some embodiments, the lower wall 1396 and/or upperwall 1398 may be defined as a locking wall configured to engage the atleast one retaining member 1333 of the retainer 1332.

With reference to FIGS. 277-306, the gripping portion 1380 of theplunger 1310, which may be sealed within the separation container 1300,may be manipulated through the flexible sealing member 1325 to preventleakage or contamination. In such embodiments, the gripping portion 1380and second distal end 1319 of the plunger 1310 may extend at leastpartially into a cavity of the flexible sealing member 1325 via an openend at the flange 1381 of the flexible sealing member. The flexiblesealing member 1325 may be made of an elastomeric material, and in someembodiments, the flexible sealing member 1325 may be made of an at leastpartially transparent material. In some embodiments, the flexiblesealing member 1325 may be retained by a cap 1330 that is structured andfunctions substantially the same as the caps (e.g., caps 130, 1030,1230) described herein, with an opening 1331 (shown in FIG. 277) throughwhich the flexible sealing member 1325 may extend. The cap 1330 may thenthread onto the body 1305 to secure a flange 1381 of the flexiblesealing member therebetween such that the flange 1381 may engage thebody and seal the second distal end 1307 of the body 1305 to close theinternal chamber 1311 of the body 1305.

With reference to FIGS. 289-295, the flexible sealing member 1325 mayinclude a top 1382, which the user may depress to also depress theplunger 1310 (shown in FIG. 287). The top 1382 and flange 1381 may beconnected by a wall 1383 of the flexible sealing member 1325. Uponactuation by the user, the top 1382 may translate downwardly towards theflange 1381 and remainder of the separation container to cause the axialmovement of the plunger 1310. The top 1382 may also be configured torotate relative to the flange 1381 during locking and unlocking of theplunger 1310 (e.g., while rotating the plunger about its longitudinalaxis) while deforming the wall 1383 therebetween such that the flange1381 remains stationary relative to the body 1305 while the top 1382rotates with the plunger 1310. The wall 1383 of the flexible sealingmember 1325 may return to a neutral position after being rotated andreleased. In some embodiments, the top 1382 may collapse slightly aroundthe user's finger during actuation. As the top 1382 and flange 1381 movecloser to each other the wall 1383 collapses and hinges to allow the top1382 to remain in contact with the plunger. In some embodiments, thewall 1383 deforms at least partially outward to allow the top 1382 todepress the plunger 1310 to a position completely flush with at least atop surface of the cap 1330. The flexible sealing member may comprise areceiving portion 1384 at the top 1382 that is configured to receive thesecond distal end of the plunger 1310 therein.

Referring to FIGS. 289-295, an embodiment of the flexible sealing member1325 is shown. In the depicted embodiment, the wall 1383 expandscontinuously outwardly when moving from the top 1382 to the flange 1381.Said differently, the flexible sealing member 1325 may define a radius r(shown in FIG. 290) in a radial direction from an axis A (also referredto as a “sealing member axis” or “displacement axis” and shown in FIG.290) extending between the top 1382 and a plane of the flange 1381. Withreference to FIG. 279, the axis A of the flexible sealing member 1325may be configured to be collinear with the longitudinal axis A of theplunger 1310, body 1305, and end cap 1350. In some embodiments, theradius at each point along the axis of the flexible sealing member 1325between the open end at the flange 1381 and the closed top 1382 isgreater than the radius at each point closer to the closed top 1382 andless than the radius at each point closer to the open end at the flange1381. In some embodiments, the wall 1383 may expand outward or bevertical when moving from the top 1382 to the flange 1381. Saiddifferently, in some embodiments, the radius at each point along theaxis of the flexible sealing member 1325 between the open end at theflange 1381 and the closed top 1382 is greater than or equal to theradius at each point closer to the closed top 1382 and less than orequal to the radius at each point closer to the open end at the flange1381. In some embodiments, the wall 1383 may have no inwardly slopedsurfaces (e.g., surfaces where the radius is less at a point closer tothe flange than another point farther from the flange). In someembodiments, the wall 1383 may have at least a two degree angle relativeto the axis A, where the angle is oriented towards the flange betweenthe wall and a vertical axis parallel to the axis A. In some embodimentseach portion of the wall 1383 may define an angle with the flange 1381that is greater than or equal to 90 degrees with respect to the shortestangle between the flange and wall when the flexible sealing member is inthe unactuated position. Such embodiments may facilitate more efficientmanufacturing and lower component costs because the flexible sealingmember 1325 may slide from its mold without stretching. In theabove-described embodiments, the angle of the wall 1383 may change uponactuation of the flexible sealing member either to rotate or axiallydepress the plunger 1310.

With continued reference to FIGS. 289-295, in some embodiments, the wall1383 may comprise a series of wall segments extending from the top 1382to the flange 1381, which vary in angle relative to the axis A. In someembodiments, as depicted in FIG. 290, the wall 1383 may alternate in itsangle between steeper and shallower angles at each junction betweensegments. With reference to FIGS. 293-295, the wall 1383 may have one ormore hinge points 1385 having a lesser wall thickness than the wallsegments on either side. These hinge points 1385 may facilitate theflexible sealing member 1325 collapsing during actuation of the plungersuch that the wall 1383 deflects out of the path of the plunger 1310. Insome embodiments, the travel distance of the plunger 1310 and thedeformation range of the flexible sealing member 1325 may be greaterthan an equivalently tall bellows design.

In the embodiment shown in FIGS. 289-291, the top 1382 is connected to afirst vertical wall segment 1386. The first vertical wall segment 1386is then connected to a first non-vertical wall segment 1387 that may beangled downwardly and outwardly from the top. The first non-verticalwall segment 1387 may then be connected to a second vertical wallsegment 1386. The second vertical wall segment 1386 may then beconnected to a second non-vertical wall segment 1387 that may be angleddownwardly and outwardly from the second vertical wall segment 1386. Thesecond non-vertical wall segment 1387 may then be connected to a thirdvertical wall segment 1386, which may abut the flange 1381. In someembodiments, a surface of the non-vertical wall segments 1387 mayinclude a vertical portion therein to define a portion of the hingepoint 1385 (e.g., portions of the outer surface or inner surface maynarrow to create a portion of a narrowed hinge point in the otherwisenon-vertical wall segment. In some embodiments, the wall segments maysmoothly transition between each other. In some embodiments, theflexible sealing member (e.g., flexible sealing members 125, 1025, 1225)of any other embodiment may be used in place of the flexible sealingmember 1325.

During downward actuation, the flexible sealing member 1325 may collapseso that the vertical wall segments 1386 translate down as thenon-vertical wall segments 1387 behave as in a hinging action (e.g., viahinge points 1385) so that in the collapsed state the segments form anested set of concentric walls. When fully compressed, the vertical wallsegments 1386 may form concentric nested ridges with the angled wallsegments (e.g., non-vertical wall segments 1387) changing orientation atthe hinge points 1385.

Referring back to FIGS. 277, 279, 284, 287, and 335-338, the separationcontainer 1300 may include a rheological control member 1342, which maybe structured and operate according to any of the embodiments discussedherein. For example, in some embodiments, the rheological control member1342, body 1305, and plunger 1310 may be structured and cooperate insubstantially the same manner as the rheological control members 1042,1242, bodies 1005, 1205, and plungers 1010, 1210 herein, and may includethe gasket 1043 and corresponding structure and operation describedtherewith. In some other embodiments, the rheological control member1342 may be structured and operate as shown and described with respectto the embodiments of FIGS. 14-31, FIGS. 32-49, or FIGS. 50-67. In someembodiments, the rheological control member 1342 may be configured toimpinge the wall 1309 of the body 1305 during loading of the sample, andthe wall 1309 may flex outwardly during centrifugation to release therheological control member. Likewise, in some embodiments, the wall 1309of the body 1305 may include a shoulder (e.g., annular shoulder 1061)that impinges the rheological control member below a certain axialposition in accordance with the embodiments described herein.

With reference to FIGS. 335-338, in some embodiments, the rheologicalcontrol member 1342 may include an annular wall 1391 comprising an innersurface 1392 defining a bore of the rheological control member throughwhich the plunger 1310 is configured to pass. The rheological controlmember 1342 may further comprise an outer surface 1393 defining an outerdiameter of the rheological control member. In some embodiments, therheological control member 1342 may further comprise one or more ribs1394. The ribs 1394 may be configured to engage the wall 1309 of thebody 1305 (e.g., at the tapered region 1303) to allow fluid to flow pastthe rheological control member 1342 and prevent the rheological memberfrom completely sealing the upper portion of the internal chamber 1311from the lower portion of the internal chamber. In some embodiments, therheological control member 1342 may comprise at least one annular trough1395 extending circumferentially about a top of the rheological controlmember. In some embodiments, fluid passes around the outer surface 1393of the rheological control member 1342 and/or through the center (e.g.,along the inner surface 1391) between the plunger 1310 and therheological control member 1342 during addition of the sample to layerthe sample (e.g., without disturbing a density cushion). In someembodiments, the annular trough 1395 may be solid and may not allowfluid to pass therethrough. The trough 1395 may be configured to captureunwanted portions of the blood culture sample (e.g., excess resinbeads). In some embodiments, the annular trough may include a hole, aplurality of holes, or a mesh screen.

Unless otherwise stated, the separation container 1300 may operate inthe same manner, may have the same properties, and may be made with thematerials and configurations of any embodiment described herein. Theseparation container 1300 of the seventh embodiment depicts anon-buoyant plunger 1310 that is retained by a retainer 1332 asdescribed above. The depicted separation container 1300 also includes arheological control member 1342 as described herein. In addition, thedepicted separation container 1300 may engage an end cap 1350 forsecuring the seal 1320 (e.g., a foil sheet or other sealing membrane) tothe body 1305 and supporting the separation container duringcentrifugation. The separation container 1300 may further include acoupling member 1340 for coupling the body 1305 with a sample collectingvessel 135, 1038, which may be used in a similar manner to the samplecollecting vessels 135, 1038 described herein.

During assembly, the separation container 1300 may be assembled in thefollowing ordered steps, some of which may be omitted depending on thefinal structure and contents of the separation container in accordancewith the embodiments described herein: (1) add the density cushion tothe internal chamber 1311; (2) connect the plunger 1310 with a retainer1332; (3) insert the rheological control member 1342 onto the plunger1310; (4) slide the gasket 1043 onto the plunger 1310 beneath therheological control member 1342 (for embodiments using a gasket); (5)engage the retainer 1332 with the supporting projections 1365 in thebody 1305, while also forming an interference or slip fit between therheological control member 1342 and the body 1305; (6) insert theflexible sealing member 1325 into the opening 1331 in the cap 1330; and(7) secure the flexible sealing member 1325 and cap 1330 onto the body1305 while encapsulating the plunger within the separation container(e.g., seal the separation container either before or after inserting asample for testing). An interference fit may be created by insertingrods into the gaps between the retainer 1332 and the plunger 1310 topress the rheological control member 1342 downwardly. In someembodiments the seal 1320 may be attached to the body 1305 prior toadding the density cushion. In accordance with some embodimentsdiscussed herein, the rheological control member may be initiallyinserted with an interference fit before being released duringcentrifugation by the outward deformation of the wall 1309 of the body1305.

During testing, the separation container 1300 may be operated in thefollowing ordered steps: (1) To add the lysed sample to the tube (e.g.,lysed from a raw sample), the cap 1330 and flexible sealing member 1325may be removed; (2) the sample may then be transferred to the body 1305,with the rheological control member 1342 preventing bulk mixing of thesample with the density cushion by being positioned between the twofluids; (3) the separation container may be optionally pre-spun beforeadding the sample to move any density cushion that may have migrated tothe top chamber during shipping or storage. The rheological controlmember 1342 may be constructed for a small clearance between it and thewall 1309 of the body 1305 and may include annular shoulders (e.g.,shoulders 1061, 1062) when not being centrifuged as discussed above(e.g., to limit the downward movement of the rheological controlmember). In some embodiments, the rheological control member 1342 mayalso be constructed so that it does not interact with or reach thetapered region 1303 of the body 1305. During centrifugation, the samplemay run down the wall 1309 of the tapered region 1303 and collect intothe pellet in the pellet region 1304.

In some embodiments, the included angle of the wall 1309 of the body1305 at the tapered region 1303 is preferably 40 degrees or less. Insome embodiments, the angle between the wall 1309 of the body 1305 and alongitudinal axis of the body 1305 at the tapered region is preferably20 degrees or less. In some embodiments, included angle of the wall 1309of the body 1305 at the tapered region 1303 is preferably from 10degrees to 40 degrees. In some embodiments, the angle between the wall1309 of the body 1305 and a longitudinal axis of the body 105 at thetapered region is preferably 5 degrees to 20 degrees. In someembodiments, the wall 1309 at the tapered region 1303 may define anincluded angle from about 20 to about 70 degrees. In some embodiments,the wall 1309 at the tapered region 1303 may define an included anglefrom 40 degrees to 60 degrees. In some embodiments, the wall 1309 at thetapered region 1303 may define an included angle from 5 degrees to 20degrees. In some embodiments, the wall 1309 at the tapered region 1303may define an included angle from 5 degrees to 60 degrees. In someembodiments, the wall 1309 at the tapered region 1303 may define anincluded angle from 5 degrees to 40 degrees. In some embodiments, thewall 1309 at the tapered region 1303 may define an included angle from10 degrees to 40 degrees. In some embodiments, the wall 1309 at thetapered region 1303 may define an included angle from 15 degrees to 40degrees. In some embodiments, the wall 1309 at the tapered region 1303may define an included angle from 20 degrees to 40 degrees. In someembodiments, the wall 1309 at the tapered region 1303 may define anincluded angle from 20 degrees to 60 degrees. In some embodiments, thewall 1309 at the tapered region 1303 may define an included angle from25 degrees to 60 degrees. In some embodiments, the wall 1309 at thetapered region 1303 may define an included angle from 30 degrees to 60degrees. In some embodiments, the wall 1309 at the tapered region 1303may define an included angle from 35 degrees to 60 degrees. In someembodiments, the wall 1309 at the tapered region 1303 may define anincluded angle from 35 degrees to 40 degrees.

In some embodiments, the wall 1309 at the tapered region 1303 may definea maximum included angle of 60 degrees. In some embodiments, the wall1309 at the tapered region 1303 may define a maximum included angle of55 degrees. In some embodiments, the wall 1309 at the tapered region1303 may define a maximum included angle of 50 degrees. In someembodiments, the wall 1309 at the tapered region 1303 may define amaximum included angle of 45 degrees. In some embodiments, the wall 1309at the tapered region 1303 may define a maximum included angle of 40degrees. In some embodiments, the wall 1309 at the tapered region 1303may define a maximum included angle of 35 degrees. In some embodiments,the wall 1309 at the tapered region 1303 may define a maximum includedangle of 30 degrees. In some embodiments, the wall 1309 at the taperedregion 1303 may define a maximum included angle of 25 degrees. In someembodiments, the wall 1309 at the tapered region 1303 may define amaximum included angle of 20 degrees.

In some embodiments, the wall 1309 at the tapered region 1303 may definea minimum included angle of 5 degrees. In some embodiments, the wall1309 at the tapered region 1303 may define a minimum included angle of10 degrees. In some embodiments, the wall 1309 at the tapered region1303 may define a minimum included angle of 15 degrees. In someembodiments, the wall 1309 at the tapered region 1303 may define aminimum included angle of 20 degrees. In some embodiments, the wall 1309at the tapered region 1303 may define a minimum included angle of 25degrees. In some embodiments, the wall 1309 at the tapered region 1303may define a minimum included angle of 30 degrees. In some embodiments,the wall 1309 at the tapered region 1303 may define a minimum includedangle of 35 degrees. In some embodiments, the wall 1309 at the taperedregion 1303 may define a minimum included angle of 40 degrees.

In some embodiments, exceeding a maximum angle may cause biomass toadhere to the wall 1309. For example, in some embodiments, the includedangle of the wall 1309 of the tapered region being over 40 degrees maycause biomass accumulation. In some embodiments, the included angle ofthe wall 1309 at the tapered region 1303 may be configured to ensurebiomass does not adhere to the wall during centrifugation, maximize thesample volume, and/or limit the length of the separation container 1300to fit within a standard syringe.

The testing may further include: (4) during addition of the sample therheological control member 1342 may float if optionally not interferencefit, while still providing mixing protection. The rheological controlmember 1342 may also have a recessed ring in the top portion, which cantrap any settling components such as resin or other particulates. Theseparticles may stay in the recessed area as the layering aid floats upthe tube either during sample addition or centrifugation. The testingmay further include: (5) after the sample is added, the cap 1330 andflexible sealing member 1325 may be placed back on the tube; and (6) theentire separation container 1300 may be placed in the centrifuge andspun. In embodiments where the rheological control member 1342 is sealedagainst the body 1305 and plunger 1310, the sample may be added directlyto an internal chamber 1311 with a lytic agent and the entire separationcontainer 1300 can be vortexed to lyse the sample before thecentrifugation step.

In operation, the process for expressing the pellet is the sameactuation and foil piercing process described herein with respect toeach other plunger embodiment, and in embodiments using a retainer 1332,an additional step of applying horizontal, rotational pressure to thegripping portion 1380 of the plunger 1310 may be used to disconnect theplunger from the retainer. With reference to FIGS. 278-282, theseparation container 1300 is shown in a storage or loaded position(depending on whether sample has been introduced to the body 1305) inwhich the plunger 1310 is attached to and retained by the retainer 1332.FIGS. 283-285 show the separation container 1300 whose plunger 1310 hasbeen released from the retainer 1332, but the plunger has not yetpierced the seal 1320. In the position shown in FIGS. 283-285, thesealing rib 1372 has abutted the wall 1309 of the body 1305 at thejunction between the tapered region 1303 and the pellet region 1304 whenthe point 1317 contacts the seal 1320, but the plunger seal 1390 has notyet engaged the pellet region 1304. In some embodiments, the plungerseal 1390 may engage the pellet region 1304 before the seal 1320 isopened. In some embodiments, the plunger seal 1390 may engage the pelletregion 1304 simultaneous with the seal 1320 is opened. In someembodiments, the plunger seal 1390 may engage the pellet region 1304after the seal 1320 is opened, and in such embodiments, the sealing rib1372 may engage the pellet region 1304 simultaneous with or prior to theseal 1320 opening (e.g., as shown in FIG. 285) to initially provide anat least partial seal of the pellet region prior to engagement of theplunger seal 1390 with the pellet region. In some embodiments, prior tothe seal 1320 opening, any density cushion present above the pelletregion 1304 may be displaced upward as the plunger 1310 moves in adownward direction along its longitudinal axis. In some instances,residual volumes of the density cushion present in the pellet region maynot affect testing performance.

Turning to FIGS. 286-288, the plunger 1310 is shown fully depressed withthe point 1317 and rib 1372 having passed through the opening at thedistal end 1306 of the body 1305, the pellet having been fullyexpressed, and the plunger seal 1390 preventing leakage of the fluid(e.g., density cushion) thereabove. For example, in some embodiments,the distance between the sealing surfaces of the rib 1372 and theplunger seal 1390 may be less than the length of the pellet region 1304to prevent leakage of the density cushion or contamination of thesample. Unless otherwise stated, features having the same referencenumeral, name, structure, or purpose in the assembly may be interchangedwith one another in any of the embodiments described herein and thepresent inventors specifically contemplate each possible permutation ofstructures and features.

In each of the above-described embodiments, once the separatedmicroorganism sample has been prepared, a subsequent interrogation stepcan be carried out to provide measurements useful for characterizationand/or identification of the microorganism. Useful interrogation meansare known in the art.

Example 1: Direct ID and AST Using Large Volume Separation Containers

In some embodiments, the separation containers (e.g., separationcontainer 100) may facilitate larger sample sizes than traditionalseparation equipment. To explore the potential of the aforementionedseparation container and centrifuge assemblies, several devices andsamples were tested in accordance with the embodiments discussed herein.In creating the instant example and the separation containers disclosedherein, the inventors noted that smaller volume devices wereinsufficient for certain species of microorganism, and as such, a largervolume separation container was designed that utilized the plunger 100,115 of the present disclosure. In these instances, the recovered biomass(i.e., number of microbial cells) was not sufficient for some species,such as A. baumannii and P. aeruginosa, so a tube with a higher volumecapacity was needed. The separation container used in the Exampleincluded substantially the same structure and operation as theseparation container 100 described herein. In practice, the mechanicalrecovery of the pellet according to the instant design provided asubstantial improvement in both ease of use and in user safety.

During the Example, four bacteria with differing pellet consistency weretested in accordance with TABLE 1 below:

TABLE 1 Species Strain Reason for Testing P. aeruginosa 1935 Small, firmbut sticky pellet (low biomass) K. pneumoniae 79382 Large, loose mucoidpellet (low density organism) E. faecium 50215 Large, moderateconsistency pellet S. aureus 60570 Firmly packed pellet, difficult todislodge

The following testing method was utilized: (1) remove a 2 mL sample ofBC broth and add to a Lysis Tube containing 4 mL LB16 buffer; (2) vortexLysis Tube and leave for 1 min at RT; (3) add 6 mL of the resultantlysate to a large scale separation container containing 2 mL of densitycushion and approx. 1 mL of polypropylene balls as a rheological controlmember (also referred to as a layering aid); (4) apply cap and spin tubefor 10 minutes at 3,000 g at RT; (5) attach a sample collecting vessel(e.g., sample collecting vessel 135) containing 1.0 mL VITEK saline tothe lower end of the body; (6) depress the plunger (e.g., plunger 110,115) to eject the pellet into the sample collecting vessel; (7) vortexor shake the tube to create a microbial suspension; (8) read theMcFarland and adjust the solution to 0.50-0.63 McFarland; (9) load thediluted suspension into appropriate VITEK AST cards; and (10) spotsuspension onto MALDI slide on a 60 C heater block, dry, add matrix, dryand load into a VITEK MS MALDI-TOF system.

The testing was performed with no leakage, provided a consistent andeasy mechanical transfer of the microbial pellets to the samplecollecting vessel, and recovered sufficient viable bacterial cells fordownstream analysis. All four bacteria were identified by MALDI-TOF (SeeFIG. 158). The test suspension VITEK2 AST card results were concordantwith historical colony AST results, all results being within EssentialAgreement, or within 1 doubling dilution of the control MIC (See FIGS.159-160).

Example 2: General Sample Processing

In some example embodiments a sample may be processed using thefollowing steps: (1) remove a cultured sample from a blood culturebottle; (2) dispense the blood culture into a lysis buffer; (3) vortexthe mixed sample to produce lysate sample; (4) add the lysate sample toa separation container; (5) centrifuge the lysate sample to generate apellet of the sample; (6) connect the separation container to a samplecollection vessel; (7) express the pellet of the sample into theseparation container; (8) resuspend the sample microorganisms in saline;and (9) dilute the resuspended sample to an appropriate concentrationfor downstream testing. In some embodiments, 2.5 mL of the culturedsample may be removed from the blood culture bottle. In someembodiments, the lysate sample may be centrifuged for 10 minutes at3,000 g. In some embodiments, the separation container may be a 5 mLseparation container.

Example 3: Yeast Processing

In an example embodiment, the various embodiments of the separationcontainer described herein may be used to process yeast samples. Oneexample yeast-processing method may include the following steps: (1)remove a cultured sample from a blood culture bottle; (2) dispense theblood culture into a lysis buffer; (3) vortex the mixed sample toproduce lysate sample; (4) add the lysate sample to a separationcontainer; (5) centrifuge the lysate sample to generate a pellet of thesample; (6) connect the separation container to a sample collectionvessel; (7) express the pellet of the sample into the separationcontainer; (8) resuspend the sample microorganisms in saline; (9) washthe resuspended sample, the washing comprising (a) centrifuging thesample, (b) removing the supernatant, (c) resuspend the sample, and (d)vortex the sample; and (10) deposit the resulting sample for downstreamtesting (e.g., MALDI-TOF ID). In some embodiments, the separationcontainer may be a small volume collection tube having a volume lessthan 5 mL.

CONCLUSION

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseembodiments of the invention pertain having the benefit of the teachingspresented in the foregoing descriptions and the associated drawings.Although some figures may or may not label certain features for ease ofviewing, a person of ordinary skill in the art may appreciate that anyfeature shown in the figures is necessarily present. Therefore, it is tobe understood that the embodiments of the invention are not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. For example, unless otherwise noted, common featuresbetween multiple embodiments may have substantially the same operationand properties. Similarly, different components (e.g., the differentrheological control members 200, 300, 400, 1042, 1242, 1342, differentplungers 110, 115, 1010, 1210, 1310 or lack thereof) may be readilysubstituted between embodiments. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

For the avoidance of doubt, the present disclosure includes the subjectmatter as defined in the following numbered paragraphs (abbreviated“para.”).

Para. 1. A separation container for extracting a portion of a sample foruse or testing, the separation container comprising:

a body defining an internal chamber, wherein the body defines anopening, and wherein the body is configured to receive the sample withinthe internal chamber;

a seal disposed across the opening, such that the seal is configured toseal the opening of the body; and

a plunger movably disposed at least partially inside the internalchamber, wherein the plunger is configured to be actuated to open theseal and express the portion of the sample.

Para. 2. The separation container of Para. 1, wherein the body definesan axis extending from the opening to a second end of the body, whereina longitudinal member of the plunger is disposed on the axis, whereinthe internal chamber defines a diameter radial to the axis, and whereinthe diameter narrows from a collection diameter to a pellet diameter ina direction extending axially from the second end to the opening.Para. 3. The separation container of Para. 2, wherein at least a portionof the plunger is configured to sealingly engage the body at a portionof the body corresponding to the pellet diameter.Para. 4. The separation container of Para. 3, wherein the at least theportion of the plunger defines a plunger diameter radial to a length ofthe longitudinal member, and wherein the plunger diameter is greaterthan or equal to the pellet diameter.Para. 5. The separation container of any one of the preceding Paras.,wherein the at least the portion of the plunger comprises a plunger sealdisposed circumferentially about the longitudinal member of the plunger,wherein the plunger seal is configured to engage the body at the portionof the body corresponding to the pellet diameter.Para. 6 The separation container of any one of the preceding Paras.,wherein the plunger is configured to allow the portion of the sample topass by the plunger from a second end towards the opening duringcentrifugation, and wherein the plunger is configured to prevent aremaining part of the sample from exiting the opening in an instance inwhich the plunger is actuated, such that the plunger is configured todivide the internal chamber into two sub-chambers.Para. 7. The separation container of any one of the preceding Paras.,wherein the plunger is buoyant in water or a density cushion material.Para. 8. The separation container of any one of the preceding Paras.,wherein the plunger defines a point at a first distal end of alongitudinal member of the plunger, and wherein the point is configuredto pierce the seal to allow fluid communication between the internalchamber and an area outside the body via the opening.Para. 9. The separation container of any one of the preceding Paras.,further comprising a sample collecting vessel configured to engage thebody, wherein the sample collecting vessel is configured to surround theopening, such that the sample collecting vessel is configured to collectthe portion of the sample passing through the seal.Para. 10. The separation container of any one of the preceding Paras.,wherein the body comprises a wall at least partially bounding theinternal chamber, and wherein the separation container furthercomprises:

a rheological control member disposed in the internal chamber, whereinthe rheological control member is disposed between the plunger and thewall, and optionally, wherein the rheological control member defines abore through which the plunger is disposed.

Para. 11. The separation container of Para. 10, wherein the body definesa second end and an axis extending from the opening to the second end,

wherein the internal chamber defines a diameter perpendicular to theaxis,

wherein the wall is at least partially flexible such that the diameterof the internal chamber is a first diameter in a static state and thediameter of the internal chamber expands to a second diameter duringcentrifugation,

wherein the rheological control member defines an outermost diameterradial to the axis of the body,

wherein the outermost diameter of the rheological control member isgreater than the first diameter, and

wherein the second diameter is greater than the outermost diameter ofthe rheological control member.

Para. 12. The separation container of Para. 11, wherein the bodycomprises a collection region defining the diameter of the internalchamber, wherein the body comprises a widened region defining a greaterdiameter than the diameter of the collection region, and wherein thegreater diameter of the widened region is greater than the outermostdiameter of the rheological control member.Para. 13. The separation container of Para. 11 or 12, wherein therheological control member comprises a second annular shouldercomprising a wide side defining the outermost diameter and a narrow sidedefining a narrower diameter than the outermost diameter.Para. 14. The separation container of any one of Paras. 10-13, whereinthe wall comprises an annular shoulder at which the diameter of theinternal chamber changes, wherein the first diameter is defined on anarrow side of the annular shoulder in the static state, and wherein theannular shoulder is configured to engage the rheological control member.Para. 15. The separation container of any one of Paras. 10 to 14,further comprising a gasket disposed circumferentially about theplunger, and wherein the gasket is configured to seal a central openingbetween the bore of the rheological control member and the plunger.Para. 16. A method for preparing samples for downstream use or testing,the method comprising:

disposing a sample into a separation container, wherein the separationcontainer comprises:

-   -   a body defining an internal chamber, wherein the body defines an        opening;    -   a seal disposed across the opening, such that the seal is        configured to seal the opening of the body; and    -   a plunger movably disposed at least partially inside the        internal chamber, wherein the plunger is configured to be        actuated to open the seal;

centrifuging the separation container to create a pellet from a portionof the sample within the internal chamber; and

expressing the pellet from the opening in the body by depressing theplunger.

Para. 17. The method for preparing samples of Para. 16, whereincentrifuging the separation container to create the pellet comprisesallowing the portion of the sample to collect at a first end of thebody, wherein the opening is defined at the first end.

Para. 18. The method for preparing samples of Para. 16 or 17, whereinexpressing the pellet comprises depressing the plunger into sealingengagement with a portion of the body to create pressure between theplunger and the seal, and expelling the pellet from the opening underthe pressure by opening the seal.Para. 19. The method for preparing samples of any one of Paras. 16-18,further comprising:

creating the sample by lysing a raw sample.

Para. 20. The method for preparing samples of any one of Paras. 16-19,further comprising:

creating the sample by culturing a raw sample.

Para. 21. The method for preparing samples of any one of Paras. 16-20,further comprising:

expressing the pellet into a sample collecting vessel.

Para. 22. The method for preparing samples of Para. 21, wherein thesample collecting vessel comprises a culture medium configured toculture organisms present in the pellet.

Para. 23. The method for preparing samples of any one of Paras. 16-22,wherein the pellet comprises viable portions of the sample suitable forantibiotic susceptibility testing (AST).

Para. 24. The method for preparing samples of any one of Paras. 16-23,wherein the pellet comprises viable portions of the sample suitable fora culture step.

Para. 25. The method for preparing samples of any one of Paras. 16-24,wherein the pellet comprises viable portions of the sample suitable forphenotypic identification methods and/or other growth-based downstreamtesting methods.

Para. 26. The method for preparing samples of any one of Paras. 16-25,wherein the pellet comprises portions of the sample suitable foridentification by mass spectrometry.

Para. 27. The method for preparing samples of any one of Paras. 16-26,further comprising analyzing the pellet using an analytical techniqueselected from a group consisting of a nucleic acid amplificationtechnique, a spectroscopy technique, an immunoassay technique, aprobe-based assay, and an agglutination test.Para. 28. A separation container comprising:

a body defining an internal chamber, wherein the body defines anopening, and wherein the body is configured to receive a sample withinthe internal chamber;

means for sealing the opening of the body; and

means for opening the means for sealing and expressing a portion of thesample.

Para. 29. The separation container of Para. 28, further comprising arheological control member configured to float on the sample; andoptionally, means for holding the rheological control member at a fixedposition during filling and releasing the rheological control member tofloat during centrifugation.Para. 30. An assembly comprising:

a retainer within a body of a separation container comprising an annularwall defining a central opening; and

a plunger comprising a longitudinal member defining a longitudinal axis;and

wherein the central opening of the retainer is configured to receive theplunger therethrough; and

wherein the retainer is configured to releasably engage the plunger tohold the plunger at a predetermined position.

Para. 31. The assembly of Para. 30, wherein the retainer furthercomprises at least one retaining member, wherein the plunger furthercomprises at least one locking member, and wherein the at least oneretaining member of the retainer is configured to releasably engage theat least one locking member of the plunger.Para. 32. The assembly of Para. 31, wherein the at least one retainingmember comprises at least one support projection extending from theannular wall, wherein the at least one retaining member is configured toengage the plunger.Para. 33. The assembly of Para. 32, wherein the annular wall defines acircumferential direction in a plane spanning the central opening andoriented about the central opening and an axial direction orientedperpendicular to the plane, and wherein the at least one supportprojection defines a circumferential wall that is longer in thecircumferential direction than in the axial direction.Para. 34. The assembly of Para. 33, wherein the at least one retainingmember further comprises at least one stop wall configured to preventrotation of the plunger in a clockwise direction or a counterclockwisedirection in an instance in which the at least one support projection isengaged with the at least one locking member of the plunger.Para. 35. The assembly of Para. 33 or 34, wherein the at least oneretaining member further comprises at least one stop wall extending froma first surface of the at least one support projection, and wherein theat least one stop wall is configured to prevent rotation of the plungerin a clockwise direction or a counterclockwise direction.Para. 36. The assembly of any one of Paras. 33 to 35, wherein the atleast one retaining member further comprises at least one locking tabextending from a first surface of the at least one support projection,and wherein the at least one locking tab is configured to engage the atleast one locking member of the plunger to releasably retain the plungerby increasing a force required to rotate the plunger about thelongitudinal axis when the at least one locking tab is engaged with theat least one locking member of the plunger.Para. 37. The assembly of any one of Paras. 33 to 36, wherein the atleast one locking member defines a C-shaped wall configured to bedisposed on both sides of the circumferential wall relative to the axialdirection in an instance in which the at least one locking member andthe at least one retaining member are engaged.Para. 38. The assembly of Para. 37, wherein the at least one retainingmember further comprises at least one locking tab extending from a firstsurface of the at least one support projection,

wherein the C-shaped wall comprises a lower wall configured to bedisposed opposite the first surface of the at least one supportprojection relative to the circumferential wall,

wherein the C-shaped wall comprises a lip configured to impinge the atleast one locking tab to at least partially resist rotation of theplunger about the longitudinal axis,

and wherein the at least one locking tab is configured to engage the atleast one locking member of the plunger to releasably retain theplunger.

Para. 39. The assembly of any one of Paras. 31 to 36, wherein the atleast one locking member comprises a locking wall extending at leastpartially perpendicular to the longitudinal axis, wherein the lockingwall is configured to engage the at least one retaining member of theretainer.Para. 40. The assembly of any one of Paras. 31 to 39, wherein the atleast one retaining member comprises at least two retaining members, andwherein the at least one locking member comprises at least two lockingmembers.Para. 41. The assembly of Para. 40, wherein the at least two retainingmembers comprise a first retaining member and a second retaining member,wherein the first retaining member is disposed diametrically oppositethe second retaining member about the annular wall,

wherein the at least two locking members comprise a first locking memberand a second locking member, and wherein the first locking member isdisposed opposite the second locking member relative to the longitudinalmember.

Para. 42. The assembly of any one of Paras. 30 to 41, wherein theplunger is configured to rotate about the longitudinal axis to engagethe retainer, and wherein in an instance in which the plunger and theretainer are engaged, the plunger is prevented from moving along thelongitudinal axis.Para. 43. The assembly of any one of Paras. 30 to 42, wherein theannular wall defines a circumferential direction in a plane spanning thecentral opening and oriented about the central opening and an axialdirection oriented perpendicular to the plane, wherein the axialdirection is parallel to the axial direction, and wherein the plunger isconfigured to rotate within the plane to engage the retainer.Para. 44. The assembly of any one of Paras. 30 to 43, wherein the bodyof the separation container defines an internal chamber, wherein theretainer is positioned within the internal chamber, wherein the plungeris configured to extend at least partially into the internal chamber,and wherein the retainer is configured to retain the plunger at thepredetermined position with respect to the body.Para. 45. The assembly of Para. 44, wherein in an instance in which theplunger and the retainer are engaged, centrifugation of the assembly isconfigured to apply centripetal force along the longitudinal axis.Para. 46. The assembly of Para. 44 or 45, wherein the retainer furthercomprises at least one retaining member, wherein the plunger furthercomprises at least one locking member,

wherein the at least one retaining member of the retainer is configuredto engage the at least one locking member of the plunger, and

wherein the at least one retaining member is configured to engage the atleast one locking member only from a rotational direction about thelongitudinal axis.

Para. 47. The assembly of any one of Paras. 44 to 46, wherein theretainer and the body of the separation container are integrally formedas a single piece.

Para. 48. The assembly of any one of Paras. 44 to 47, wherein theretainer is separately connected to the body of the separationcontainer.

Para. 49. The assembly of Para. 48, wherein the retainer is press fitinto the body of the separation container.

Para. 50. The assembly of any one of Paras. 44 to 49, furthercomprising:

a flexible sealing member disposed at a second end of the body of theseparation container, wherein a second distal end of the plunger isconfigured to extend at least partially into the flexible sealingmember; and

a cap threaded to the body at the second end, wherein a portion of theflexible sealing member is configured to be disposed between the cap andthe body, and wherein the cap defines an opening through which a secondportion of the flexible sealing member and the second distal end of theplunger are configured to extend; and

wherein the plunger is configured to disengage from the retainer bygripping and rotating the plunger through the flexible sealing member.

Para. 51. A method of operating a plunger and retainer assembly, theassembly comprising a retainer within a body of a separation containercomprising an annular wall defining a central opening, at least oneretaining member, and a plunger comprising a longitudinal memberdefining a longitudinal axis; the method comprising:

rotating the plunger about the longitudinal axis to disengage theplunger from the retainer; and

actuating the plunger by applying a force to the plunger along thelongitudinal axis.

Para. 52. The method of Para. 51, wherein the plunger is at leastpartially disposed in the body of the separation container, the methodfurther comprising:

disposing a sample in the body of the separation container;

centrifuging the assembly before the rotating and the actuating of theplunger, such that the plunger is configured to be retained at apredetermined position relative to the body of the separation containerduring centrifugation.

Para. 53. The method of Para. 52 further comprising lysing the samplebefore disposing the sample in the body of the separation container.

Para. 54. An assembly comprising:

a plunger comprising a longitudinal member defining a longitudinal axis;

a body of a separation container; and

means for releasably retaining the plunger at least partially within thebody of the separation container at a predetermined position.

Para. 55. The assembly of Para. 54, wherein the means for releasablyretaining the plunger define an engaged state and a disengaged state,and wherein in the engaged state, the means for releasably retaining theplunger are configured to prohibit the plunger from moving along thelongitudinal axis.Para. 56. The assembly of Para. 55, wherein the means for releasablyretaining the plunger are configured to engage and disengage by rotatingthe plunger about the longitudinal axis.Para. 57. A plunger for expressing a portion of a sample from acontainer, the plunger comprising:

a longitudinal member defining a first distal end, a second distal end,and an axis extending between the first distal end and the second distalend; and

a plunger seal positioned about the longitudinal member at a locationbetween the first distal end of the longitudinal member and the seconddistal end of the longitudinal member, the plunger seal defining aplunger seal diameter perpendicular to the axis; and

wherein at least a portion of the longitudinal member between thelocation and the first distal end of the longitudinal member defines aplunger diameter that is less than the plunger seal diameter, such thatin operation the at least the portion of the longitudinal member isconfigured to retain a volume of sample between a wall of the container,a distal end of the container, the plunger seal, and the at least theportion of the longitudinal member.

Para. 58. The plunger of Para. 57, wherein the first distal end of thelongitudinal member defines a point configured to pierce a seal of thecontainer.

Para. 59. The plunger of Para. 57 or 58, wherein the plunger seal isovermolded onto the longitudinal member.

Para. 60. The plunger of any one of the preceding claims, wherein thelongitudinal member defines a through passage extending through thelongitudinal member at least partially perpendicular to the axis,wherein the plunger seal is disposed on the longitudinal member at anaxial location of the through passage, and wherein the plunger sealextends through the through passage.Para. 61. The plunger of any one of Paras. 57 to 60, wherein the plungerseal is elastomeric.Para. 62. The plunger of Para. 57 or 58, wherein the plunger seal isintegral with the longitudinal member.Para. 63. The plunger of any one of Paras. 57 to 62, further comprisinga sealing rib disposed circumferentially about the longitudinal memberof the plunger, wherein the sealing rib is disposed between the plungerseal and the first distal end relative to the axis.Para. 64. The plunger of any one of Paras. 57 to 62, wherein thelongitudinal member defines a shoulder between the second distal end andthe plunger seal, wherein a diameter of the shoulder is greater than theplunger seal diameter.Para. 65. A separation container comprising:

a container body defining an internal chamber configured to receive asample, wherein the container body defines an opening, a collectionregion having a collection diameter, and a pellet region having a pelletdiameter, wherein the collection diameter is greater than the pelletdiameter, and wherein the pellet region is defined between the openingand the collection region in the internal chamber;

a seal disposed across the opening, such that the seal is configured toseal the opening of the container body; and

a plunger configured to be disposed at least partially within theinternal chamber to open the seal, the plunger comprising:

-   -   a longitudinal member defining a first distal end, a second        distal end, and an axis extending between the first distal end        and the second distal end, wherein the first distal end of the        plunger is configured to open the seal; and    -   a plunger seal positioned about the longitudinal member at a        location between the first distal end of the longitudinal member        and the second distal end of the longitudinal member, the        plunger seal defining a plunger seal diameter perpendicular to        the axis;

wherein the plunger seal diameter is greater than or equal to the pelletdiameter; and

wherein at least a portion of the longitudinal member between thelocation and the first distal end of the longitudinal member defines aplunger diameter that is less than the plunger seal diameter, such thatin operation the at least the portion of the longitudinal member isconfigured to retain a volume between a wall of the container, a distalend of the container, the plunger seal, and the at least the portion ofthe longitudinal member.

Para. 66. The separation container of Para. 65, wherein the longitudinalmember defines a shoulder between the second distal end and the plungerseal;

wherein a diameter of the shoulder is greater than the pellet diameter,and less than the collection diameter; and

wherein the shoulder is configured to impinge the container body todefine a maximum displacement of the plunger.

Para. 67. The separation container of Para. 66, wherein an axialdistance between the plunger seal and the shoulder is less than or equalto an axial length of the pellet region, such that the plunger sealremains at least partially within the container body at the maximumdisplacement of the plunger.Para. 68. The separation container of Para. 66 or 67, wherein an axialdistance between the plunger seal and the first distal end of theplunger is less than or equal to an axial length of the pellet regionsuch the plunger seal is configured to engage the pellet region of thecontainer body before the plunger opens the seal during actuation.Para. 69. The separation container of any one of Paras. 66 to 68,wherein the first distal end of the longitudinal member defines a pointconfigured to pierce the seal.Para. 70. The separation container of any one of Paras. 66 to 68,wherein an axial distance between the plunger seal and the shoulder andan axial length of the pellet region are configured such that theplunger seal remains at least partially within the container body at themaximum displacement of the plunger.Para. 71. The separation container of any one of Paras. 65 to 70,wherein the plunger seal is overmolded onto the longitudinal member.Para. 72. The separation container of any one of Paras. 65 to 71,wherein the container body further comprises a tapered region connectingthe collection region and the pellet region, and wherein a diameter ofthe tapered region varies relative to an axial direction from the pelletdiameter at a junction between the pellet region and the tapered regionto the collection diameter at a junction between the collection regionand the tapered region.Para. 73. A method of expressing a portion of a sample from theseparation container, the separation container comprising a containerbody defining an internal chamber, an opening, a collection regionhaving a collection diameter, and a pellet region having a pelletdiameter, wherein the collection diameter is greater than the pelletdiameter, and wherein the pellet region is defined between the openingand the collection region in the internal chamber; a seal disposedacross the opening; and a plunger disposed at least partially within theinternal chamber, the plunger comprising a longitudinal member defininga first distal end, a second distal end, and an axis extending betweenthe first distal end and the second distal end, and a plunger sealpositioned about the longitudinal member at a location between the firstdistal end of the longitudinal member and the second distal end of thelongitudinal member, wherein the plunger seal defines a plunger sealdiameter perpendicular to the axis; wherein the plunger seal diameter isgreater than or equal to the pellet diameter; and wherein at least aportion of the longitudinal member between the location and the firstdistal end of the longitudinal member defines a plunger diameter that isless than the plunger seal diameter; the method comprising:

displacing the plunger along the axis to cause the plunger seal toengage the container body at the pellet region, wherein engagement ofthe plunger seal and the container body retains the volume between awall of the container, a distal end of the container, the plunger seal,and the at least the portion of the longitudinal member.

further displacing the plunger along the axis to open the seal with thefirst distal end of the plunger; and

further displacing the plunger to express the volume of the sample fromthe opening via pressure created by the plunger seal.

Para. 74. A separation container comprising:

a container body defining an internal chamber configured to receive asample, wherein the container body defines an opening, a collectionregion having a collection diameter, and a pellet region having a pelletdiameter, wherein the collection diameter is greater than the pelletdiameter, and wherein the pellet region is defined between the openingand the collection region in the internal chamber;

means for sealing the opening;

means for opening the means for sealing the opening and expressing aportion of the sample; and

means for fluidically separating the portion of the sample from otherfluid in the internal chamber during expression of the portion of thesample.

Para. 75. The separation container of Para. 74, wherein the means forfluidically separating the portion of the sample from the other fluid inthe internal chamber is attached to the means for opening the means forsealing the opening and expressing the portion of the sample.Para. 76. The separation container of Para. 74 or 75, wherein the meansfor opening the means for sealing and expressing the portion of thesample is configured to express only the portion of the sample duringoperation.Para. 77. An assembly comprising:

a separation container comprising:

-   -   a body, the body defining an internal chamber, wherein the body        defines an opening at a first end, and wherein the body is        configured to receive a sample within the internal chamber; and    -   a seal attached to the body and disposed across the opening,        wherein the seal is configured to seal the opening of the body;        and

an end cap disposed at the first end of the body and removably connectedto the body, wherein the seal is positioned between the end cap and thebody.

Para. 78. The assembly of Para. 77, wherein the body further defines anannular surface around the opening, and wherein the seal is attached tothe annular surface.

Para. 79. The assembly of Para. 78, wherein the end cap defines asupport surface configured to be positioned parallel to the annularsurface of the body with the seal being disposed between the supportsurface and the annular surface.

Para. 80. The assembly of any one of Paras. 77 to 79, wherein the sealcomprises a membrane.

Para. 81. The assembly of Para. 80, wherein the seal comprises a foilsheet.

Para. 82. The assembly of any one of Paras. 77 to 81, wherein the sealis welded to the body of the separation container.

Para. 83. The assembly of any one of Paras. 77 to 82, wherein the endcap is a centrifuge adaptor comprising at least one of a flat distal endand a tapered portion configured to engage a centrifuge cup.

Para. 84. The assembly of Para. 83, wherein the end cap comprises both aflat distal end and a tapered portion.

Para. 85. The assembly of Para. 84, wherein the flat distal endcomprises a recess configured to receive a post therein.

Para. 86. The assembly of any one of Paras. 77 to 85, wherein the firstend of the body is inserted into a cavity of the end cap, and whereinthe end cap is removably connected to the body by friction.

Para. 87. The assembly of Para. 86, further comprising a coupling memberdisposed between the end cap and the container body, wherein the end capcomprises an inner surface configured to apply a radially inwardpressure to the coupling member and body to retain the end cap on thebody.Para. 88. The assembly of Para. 87, wherein the coupling membercomprises an elastomeric sleeve disposed over the first end of the bodybetween the body and the end cap.Para. 89. The assembly of Para. 88 further comprising a samplecollecting vessel; wherein the end cap is configured to be removable;wherein in an instance in which the end cap is removed, the samplecollecting vessel is configured to fit over and be retained by at leastthe elastomeric sleeve.Para. 90. The assembly of Para. 89 further comprising a plungercomprising a longitudinal member defining a first distal end, a seconddistal end, and an axis extending between the first distal end and thesecond distal end; wherein the plunger is configured to open the sealand express at least a portion of the sample from the opening of thebody of the separation container; and wherein the elastomeric sleeve isconfigured to seal the sample collecting vessel from an externalenvironment.Para. 91. The assembly of any one of Paras. 88 to 90, wherein the endcap further comprises one or more ribs configured to compress theelastomeric sleeve to release air trapped in the end cap duringinsertion of the end cap onto the body.Para. 92. The assembly of any one of Paras. 88 to 91, wherein theelastomeric sleeve is disposed between the first end and the second endof the body, such that the end cap is configured to directly contact theseal.Para. 93. A method using an assembly, the assembly comprising aseparation container comprising a body, the body defining an internalchamber, wherein the body defines an opening at a first end, and whereinthe body is configured to receive a sample within the internal chamber,the separation container further comprising a seal attached to the bodyand disposed across the opening, wherein the seal is configured to sealthe opening of the body; the assembly further comprising an end cap; themethod comprising:

releasably engaging the end cap with the body such that the seal isdisposed between the end cap and the body, wherein the end cap isconfigured to prevent the seal from detaching from the body duringcentrifugation.

Para. 94. The method of Para. 93 further comprising:

centrifuging the assembly;

disengaging the end cap from the body; and

opening the seal.

Para. 95. The method of Para. 94, wherein the assembly further comprisesa sample collecting vessel, the method further comprising:

engaging the sample collecting vessel with the body at the opening; and

expressing at least a portion of the sample into the sample collectingvessel.

Para. 96. The method of Para. 95, wherein the assembly further comprisesa plunger comprising a longitudinal member defining a first distal end,a second distal end, and an axis extending between the first distal endand the second distal end, wherein opening the seal comprises openingthe seal with the first distal end of the plunger, wherein expressingthe pellet comprises depressing the plunger into sealing engagement witha portion of the body to create pressure between the plunger and theseal and expelling the pellet from the opening under the pressure.Para. 97. An assembly comprising:

a separation container comprising:

-   -   a body, the body defining an internal chamber, wherein the body        defines an opening at a first end, and wherein the body is        configured to receive a sample within the internal chamber; and    -   means for sealing the opening of the body; and

means for preventing the seal from separating from the body duringcentrifugation.

Para. 98. The assembly of Para. 97, wherein the means for sealing theopening of the body is attached to the body.

Para. 99. The assembly of Para. 97 or 98, wherein the means forpreventing the seal from separating from the body during centrifugationis frictionally retained against the seal.

Para. 100. The assembly of any one of Paras. 97 to 99, wherein the meansfor preventing the seal from separating from the body duringcentrifugation is configured to apply a retaining force to the means forsealing the opening of the body during centrifugation.Para. 101. The assembly of any one of Paras. 97 to 100, wherein themeans for preventing the seal from separating from the body duringcentrifugation comprises means for releasing air trapped between thebody and the means for preventing the seal from separating from the bodyduring centrifugation.Para. 102. A separation container comprising:

a body defining an internal chamber, wherein the body defines a firstopening at a first end and a second opening at a second end, and whereinthe body is configured to receive a sample within the internal chamber;

a seal disposed across the first opening, such that the seal isconfigured to seal the first opening of the body;

a plunger movably disposed at least partially inside the internalchamber, wherein the plunger is configured to be actuated to open theseal and extract a portion of the sample; and

a flexible sealing member at least partially covering the secondopening, wherein at least a portion of the plunger is configured toextend at least partially into the flexible sealing member, such thatcompression of the flexible sealing member is configured to actuate theplunger.

Para. 103. The separation container of Para. 102, wherein the flexiblesealing member comprises a flange defining an annular surface configuredto engage the body.

Para. 104. The separation container of Para. 103, wherein the flexiblesealing member further comprises a wall extending from the flange, andwherein an angle between the flange and the wall is greater than orequal to 90 degrees when the flexible sealing member is in an unactuatedposition.Para. 105. The separation container of Para. 104, wherein the wallcomprises a plurality of wall segments, and wherein an angle between theflange and each of the plurality of wall segments is greater than orequal to 90 degrees when the flexible sealing member is in theunactuated position.Para. 106. The separation container of any one of Paras. 102 to 105,further comprising a cap secured to the body at the second end, whereina portion of the flexible sealing member is configured to be disposedbetween the cap and the body, and wherein the cap defines an openingthrough which a second portion of the flexible sealing member and thesecond end of the plunger are configured to extend.Para. 107. The separation container of any one of Paras. 102 to 106,wherein the flexible sealing member comprises a bellows-shaped gasket.Para. 108. The separation container of any one of Paras. 102 to 107,wherein the flexible sealing member defines an open end configured toreceive the portion of the plunger therein, and wherein the flexiblesealing member further defines a closed end, such that the flexiblesealing member is configured to engage the body at the open end toenclose the internal chamber and a cavity of the flexible sealingmember.Para. 109. The separation container of Para. 108, wherein the plungercomprises a first end, a second end, and a longitudinal axis extendingbetween the first end and the second end,

wherein the portion of the plunger comprises the second end of theplunger,

wherein the flexible sealing member defines a sealing member axisextending between the open end and the closed end,

wherein the sealing member axis is configured to be collinear with thelongitudinal axis of the plunger in an operational position,

wherein the flexible sealing member defines a radius perpendicular tothe longitudinal axis, and

wherein the radius is configured to decrease from the open end to theclosed end.

Para. 110. The separation container of Para. 109, wherein the radius ateach point along the sealing member axis between the open end and theclosed end is less than or equal to the radius at each point closer tothe closed end and greater than or equal to the radius at each pointcloser to the open end.Para. 111. The separation container of any one of Paras. 108 to 110,wherein when the flexible sealing member is actuated, the closed end ofthe flexible sealing member is configured to move toward the open end ofthe flexible sealing member along a displacement axis.Para. 112. The separation container of Para. 111, wherein when theflexible sealing member is actuated, the closed end is configured to bepositioned at a same axial position along the displacement axis as orcloser to the first end of the body than the open end.Para. 113. The separation container of Paras. 102 to 112, wherein theflexible sealing member defines a wall configured to at least partiallysurround the portion of the plunger, wherein the wall defines aninwardly concave shape, such that the wall is configured to flexoutwardly from the plunger when the plunger is actuated.Para. 114. The separation container of Paras. 102 to 112, wherein theflexible sealing member comprises a first circumferential wall segmentconnected to a top of the flexible sealing member, a secondcircumferential wall segment connected to the first circumferential wallsegment, and a third circumferential wall segment connected to thesecond circumferential wall segment;

wherein the second circumferential wall segment is concentric about alongitudinal axis of the plunger; and

wherein the first circumferential wall segment and the secondcircumferential wall segment are each angled at least partially inwardlytowards the plunger from their respective connections to the secondcircumferential wall segment.

Para. 115. A method of using a separation container, the separationcontainer comprising a body defining an internal chamber, wherein thebody defines a first opening at a first end and a second opening at asecond end; the separation container further comprising a seal disposedacross the first opening, such that the seal is configured to seal thefirst opening of the body; a plunger movably disposed at least partiallyinside the internal chamber; and a flexible sealing member at leastpartially covering the second opening, wherein at least a portion of theplunger is configured to extend at least partially into the flexiblesealing member; the method further comprising:

disposing a sample in the internal chamber of the separation container;

centrifuging the separation container; and

after centrifugation, compressing the flexible sealing member to actuatethe plunger and open the seal to extract a portion of the sample.

Para. 116. The method of Para. 115, wherein the plunger is held axiallyby a retainer during centrifugation, wherein compressing the flexiblesealing member to actuate the plunger further comprises unlocking theplunger from the retainer.

Para. 117. The method of Para. 116, wherein compressing the flexiblesealing member to actuate the plunger further comprises rotating aportion of the flexible sealing member and the plunger about alongitudinal axis of the plunger, wherein the longitudinal axis extendsfrom a first end of the plunger to a second end of the plunger.Para. 118. The method of Para. 117, wherein rotating the portion of theflexible sealing member comprises deforming the flexible sealing membersuch that the portion of the flexible sealing member rotates while aflange of the flexible sealing member remains fixed relative to thebody.Para. 119. The method of Paras. 117 or 118, wherein the separationcontainer further comprises a cap secured to the body at the second end,

wherein a second portion of the flexible sealing member is configured tobe disposed between the cap and the body,

wherein the cap defines an opening through which the portion of theflexible sealing member and the second end of the plunger are configuredto extend.

Para. 120. A separation container comprising:

a body defining an internal chamber, wherein the body defines a firstopening at a first end and a second opening at a second end, and whereinthe body is configured to receive a sample within the internal chamber;

means for sealing the first opening of the body;

means for opening the means for sealing and expressing a portion of thesample; and

means for allowing manipulation of the means for opening the means forsealing and expressing the portion of the sample while also sealing thesecond opening of the body.

Para. 121. The separation container of Para. 120, wherein the means forallowing manipulation of the means for opening the means for sealing andexpressing the portion of the sample while also sealing the secondopening of the body is configured to allow both rotational and axialmovement of the means for sealing and expressing the portion of thesample.

The invention claimed is:
 1. A method of using a separation container,the separation container comprising a body defining an internal chamber,wherein the body defines a first opening at a first end and a secondopening at a second end; the separation container further comprising aseal disposed across the first opening, such that the seal is configuredto seal the first opening of the body; a plunger movably disposed atleast partially inside the internal chamber; and a flexible sealingmember at least partially covering the second opening, wherein at leasta portion of the plunger is configured to extend at least partially intothe flexible sealing member; the method further comprising: disposing asample in the internal chamber of the separation container; centrifugingthe separation container, wherein the plunger is held axially by aretainer during centrifugation; and after centrifugation, manipulatingthe flexible sealing member to unlock the plunger from the retainer anddepressing the flexible sealing member to actuate the plunger and openthe seal to extract a portion of the sample.
 2. The method of claim 1,wherein manipulating the flexible sealing member to unlock the plungerfrom the retainer comprises rotating a portion of the flexible sealingmember and the plunger about a longitudinal axis of the plunger, whereinthe longitudinal axis extends from a first end of the plunger to asecond end of the plunger.
 3. The method of claim 2, wherein rotatingthe portion of the flexible sealing member comprises deforming theflexible sealing member such that the portion of the flexible sealingmember rotates while a flange of the flexible sealing member remainsfixed relative to the body.
 4. The method of claim 3, wherein theflexible sealing member comprises a first circumferential wall segmentconnected to a top of the flange, a second circumferential wall segmentconnected to the first circumferential wall segment, and a thirdcircumferential wall segment connected to the second circumferentialwall segment, wherein the second circumferential wall segment isconcentric about the longitudinal axis of the plunger, and wherein thefirst circumferential wall segment and the third circumferential wallsegment are each angled at least partially inwardly towards the plungerfrom their respective connections to the second circumferential wallsegment.
 5. The method of claim 2, wherein the separation containerfurther comprises a cap secured to the body at the second end, wherein asecond portion of the flexible sealing member is configured to bedisposed between the cap and the body, wherein the cap defines anopening through which the portion of the flexible sealing member and thesecond end of the plunger are configured to extend.
 6. The method ofclaim 1, wherein the flexible sealing member is configured to deformabout a longitudinal axis during manipulation of the plunger tofacilitate rotation of the plunger, and the flexible sealing member isconfigured to deflect towards the first opening to facilitate actuationof the plunger.
 7. The method of claim 1, wherein the plunger furthercomprises one or more support members and wherein the retainer furthercomprises one or more retaining members.
 8. The method of claim 7,further comprising: before centrifugation, engaging the plunger with theretainer such that the plunger is held axially by the retainer.
 9. Themethod of claim 8, wherein the plunger with the retainer comprises:axially aligning the one or more support members of the plunger with theone or more retaining members of the retainer; and rotating the plungerabout a longitudinal axis of the plunger in a first direction until theone or more support members engage the one or more retaining members ofthe retainer and the plunger is prevented from further rotation in thefirst direction, wherein the longitudinal axis extends from a first endof the plunger to a second end of the plunger.
 10. The method of claim9, wherein manipulating the flexible sealing member to unlock theplunger from the retainer comprises: rotating the plunger about thelongitudinal axis of the plunger in a second direction opposite thefirst direction such that the one or more support members of the plungerare detached from the one or more retaining members of the retainer,thereby releasing the plunger to be axially actuated.
 11. The method ofclaim 1, wherein the flexible sealing member comprises a bellows-shapedgasket.
 12. The method of claim 1, wherein the flexible sealing memberdefines an open end configured to receive the portion of the plungertherein, and wherein the flexible sealing member further defines aclosed end, such that the flexible sealing member is configured toengage the body at the open end to enclose the internal chamber and acavity of the flexible sealing member.
 13. The method of claim 12,wherein the plunger comprises a first end, a second end, and alongitudinal axis extending between the first end and the second end,wherein the portion of the plunger comprises the second end of theplunger, wherein the flexible sealing member defines a sealing memberaxis extending between the open end and the closed end, wherein thesealing member axis is configured to be collinear with the longitudinalaxis of the plunger in an operational position, wherein the flexiblesealing member defines a radius perpendicular to the longitudinal axis,and wherein the radius is configured to decrease from the open end tothe closed end.
 14. The method of claim 13, wherein the radius at eachpoint along the sealing member axis between the open end and the closedend is less than or equal to the radius at each point closer to theclosed end and greater than or equal to the radius at each point closerto the open end.
 15. The method of claim 13, wherein when the flexiblesealing member is actuated, the closed end of the flexible sealingmember is configured to move toward the open end of the flexible sealingmember along the sealing member axis.
 16. The method of claim 15,wherein when the flexible sealing member is actuated, the closed end isconfigured to be positioned at a same axial position along the sealingmember axis as or closer to the first end of the body than the open end.17. The method of claim 1, wherein the flexible sealing member comprisesa flange defining an annular surface configured to engage the body. 18.The method of claim 17, wherein the flexible sealing member furthercomprises a wall connecting to and extending from the flange, andwherein a smallest angle between the flange and the wall, at anintersection of the flange and the wall, is greater than or equal to 90degrees when the flexible sealing member is in an unactuated position.19. The method of claim 1, wherein disposing the sample in the internalchamber comprises pouring the sample through at least one gap betweenthe plunger and the retainer.